Dual variable domain immunnoglobulins and uses thereof

ABSTRACT

The present invention relates to engineered multivalent and multispecific binding proteins, methods of making, and specifically to their uses in the prevention, diagnosis, and/or treatment of disease.

CROSS REFERENCE TO RELATED APPLICATION

This application claims priority to U.S. Provisional Application Ser.No. 61/425,671 filed on Dec. 21, 2010, the contents of which areincorporated herein.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted in ASCII format via EFS-Web and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Mar. 23, 2012, isnamed 10718USO.txt and is 132,674 bytes in size.

FIELD OF THE INVENTION

The present invention relates to multivalent and multispecific bindingproteins, methods of making, and specifically to their uses in the,diagnosis, prevention and/or treatment of acute and chronic inflammatorydiseases, cancer, and other diseases.

BACKGROUND OF THE INVENTION

Engineered proteins, such as multispecific antibodies that bind two ormore antigens are known in the art. Such multispecific binding proteinscan be generated using cell fusion, chemical conjugation, or recombinantDNA techniques.

Bispecific antibodies have been produced using quadroma technology (seeMilstein and Cuello (1983) Nature 305(5934):537-40) based on the somaticfusion of two different hybridoma cell lines expressing murinemonoclonal antibodies (mAbs) with the desired specificities of thebispecific antibody. Because of the random pairing of two differentimmunoglobulin (Ig) heavy and light chains within the resultinghybrid-hybridoma (or quadroma) cell line, up to ten different Ig speciesare generated, of which only one is the functional bispecific antibody.The presence of mis-paired by-products, and significantly reducedproduction yields, means sophisticated purification procedures arerequired.

Bispecific antibodies can also be produced by chemical conjugation oftwo different mAbs (see Staerz et al. (1985) Nature 314(6012):628-31).This approach does not yield homogeneous preparation. Other approacheshave used chemical conjugation of two different mAbs or smaller antibodyfragments (see Brennan et al. (1985) Science 229(4708):81-3).

Another method used to produce bispecific antibodies is the coupling oftwo parental antibodies with a hetero-bifunctional crosslinker, but theresulting bispecific antibodies suffer from significant molecularheterogeneity because reaction of the crosslinker with the parentalantibodies is not site-directed. To obtain more homogeneous preparationsof bispecific antibodies two different Fab fragments have beenchemically crosslinked at their hinge cysteine residues in asite-directed manner (see Glennie et al. (1987) J. Immunol.139(7):2367-75). But this method results in Fab′2 fragments, not fullIgG molecule.

A wide variety of other recombinant bispecific antibody formats havebeen developed (see Kriangkum et al. (2001) Biomol. Eng. 18(2):31-40).Amongst them tandem single-chain Fv molecules and diabodies, and variousderivatives thereof, are the most widely used. Routinely, constructionof these molecules starts from two single-chain Fv (scFv) fragments thatrecognize different antigens (see Economides et al. (2003) Nat. Med.9(1):47-52). Tandem scFv molecules (taFv) represent a straightforwardformat simply connecting the two scFv molecules with an additionalpeptide linker. The two scFv fragments present in these tandem scFvmolecules form separate folding entities. Various linkers can be used toconnect the two scFv fragments and linkers with a length of up to 63residues (see Nakanishi et al. (2001) Ann. Rev. Immunol. 19:423-74).Although the parental scFv fragments can normally be expressed insoluble form in bacteria, it is, however, often observed that tandemscFv molecules form insoluble aggregates in bacteria. Hence, refoldingprotocols or the use of mammalian expression systems are routinelyapplied to produce soluble tandem scFv molecules. In a recent study, invivo expression by transgenic rabbits and cattle of a tandem scFvdirected against CD28 and a melanoma-associated proteoglycan wasreported (see Gracie et al. (1999) J. Clin. Invest. 104(10):1393-401).In this construct, the two scFv molecules were connected by a CH1 linkerand serum concentrations of up to 100 mg/L of the bispecific antibodywere found. Various strategies including variations of the domain orderor using middle linkers with varying length or flexibility were employedto allow soluble expression in bacteria. A few studies have now reportedexpression of soluble tandem scFv molecules in bacteria (see Leung etal. (2000) J. Immunol. 164(12):6495-502; Ito et al. (2003) J. Immunol.170(9):4802-9; Karni et al. (2002) J. Neuroimmunol. 125(1-2):134-40)using either a very short Ala3 linker or long glycine/serine-richlinkers. In a recent study, phage display of a tandem scFv repertoirecontaining randomized middle linkers with a length of 3 or 6 residueswas employed to enrich for those molecules that are produced in solubleand active form in bacteria. This approach resulted in the isolation ofa tandem scFv molecule with a 6 amino acid residue linker (see Arndt andKrauss (2003) Methods Mol. Biol. 207:305-21). It is unclear whether thislinker sequence represents a general solution to the soluble expressionof tandem scFv molecules. Nevertheless, this study demonstrated thatphage display of tandem scFv molecules in combination with directedmutagenesis is a powerful tool to enrich for these molecules, which canbe expressed in bacteria in an active form.

Bispecific diabodies (Db) utilize the diabody format for expression.Diabodies are produced from scFv fragments by reducing the length of thelinker connecting the VH and VL domain to approximately 5 residues (seePeipp and Valerius (2002) Biochem. Soc. Trans. 30(4):507-11). Thisreduction of linker size facilitates dimerization of two polypeptidechains by crossover pairing of the VH and VL domains. Bispecificdiabodies are produced by expressing, two polypeptide chains with,either the structure VHA-VLB and VHB-VLA (VH-VL configuration), orVLA-VHB and VLB-VHA (VL-VH configuration) within the same cell. A largevariety of different bispecific diabodies have been produced in the pastand most of them are expressed in soluble form in bacteria. However, arecent comparative study demonstrates that the orientation of thevariable domains can influence expression and formation of activebinding sites (see Mack et al. (1995) Proc. Natl. Acad. Sci. USA92(15):7021-5). Nevertheless, soluble expression in bacteria representsan important advantage over tandem scFv molecules. However, since twodifferent polypeptide chains are expressed within a single cell inactivehomodimers can be produced together with active heterodimers. Thisnecessitates the implementation of additional purification steps inorder to obtain homogenous preparations of bispecific diabodies. Oneapproach to force the generation of bispecific diabodies is theproduction of knob-into-hole diabodies (see Holliger et al. (1993) Proc.Natl. Acad. Sci. USA 90(14):6444-8.18). This was demonstrated for abispecific diabody directed against HER2 and CD3. A large knob wasintroduced in the VH domain by exchanging Val37 with Phe and Leu45 withTrp and a complementary hole was produced in the VL domain by mutatingPhe98 to Met and Tyr87 to Ala, either in the anti-HER2 or the anti-CD3variable domains. By using this approach the production of bispecificdiabodies could be increased from 72% by the parental diabody to over90% by the knob-into-hole diabody. Importantly, production yields onlyslightly decrease as a result of these mutations. However, a reductionin antigen-binding activity was observed for several constructs. Thus,this rather elaborate approach requires the analysis of variousconstructs in order to identify those mutations that produceheterodimeric molecule with unaltered binding activity. In addition,such approach requires mutational modification of the immunoglobulinsequence at the constant region, thus creating non-native andnon-natural form of the antibody sequence, which may result in increasedimmunogenicity, poor in vivo stability, as well as undesirablepharmacokinetics.

Single-chain diabodies (scDb) represent an alternative strategy forimproving the formation of bispecific diabody-like molecules (seeHolliger and Winter (1997) Cancer Immunol. Immunother. 45(3-4):128-30;Wu et al. (1996) Immunotechnology 2(1):21-36). Bispecific single-chaindiabodies are produced by connecting the two diabody-forming polypeptidechains with an additional middle linker with a length of approximately15 amino acid residues. Consequently, all molecules with a molecularweight corresponding to monomeric single-chain diabodies (50-60 kDa) arebispecific. Several studies have demonstrated that bispecific singlechain diabodies are expressed in bacteria in soluble and active formwith the majority of purified molecules present as monomers (seeHolliger and Winter (1997) Cancer Immunol. Immunother. 45(3-4):128-30;Wu et al. (1996) Immunotechnol. 2(1):21-36; Pluckthun and Pack (1997)Immunotechnol. 3(2):83-105; Ridgway et al. (1996) Protein Engin.9(7):617-21). Thus, single-chain diabodies combine the advantages oftandem scFvs (all monomers are bispecific) and diabodies (solubleexpression in bacteria).

More recently diabodies have been fused to Fc to generate more Ig-likemolecules, named di-diabodies (see Lu et al. (2004) J. Biol. Chem.279(4):2856-65). In addition, multivalent antibody constructs comprisingtwo Fab repeats in the heavy chain of an IgG and that bind four antigenmolecules have been described (see PCT Publication No. WO 0177342, andMiller et al. (2003) J. Immunol. 170(9):4854-61).

There is a need in the art for improved multivalent binding proteinsthat bind two or more antigens. U.S. Pat. No. 7,612,181 provides a novelfamily of binding proteins that bind two or more antigens with highaffinity, and which are called dual variable domain immunoglobulins(DVD-Ig™). The present invention provides further novel binding proteinsthat bind two or more antigens.

SUMMARY OF THE INVENTION

This invention pertains to multivalent binding proteins that bind two ormore antigens. The present invention provides a novel family of bindingproteins that bind two or more antigens with high affinity.

In one embodiment the invention provides a binding protein comprising apolypeptide chain, wherein the polypeptide chain comprisesVD1-(X1)n-VD2-C-(X2)n, wherein VD1 is a first variable domain, VD2 is asecond variable domain, C is a constant domain, X1 represents an aminoacid or polypeptide, X2 represents an Fc region and n is 0 or 1. In anembodiment the VD1 and VD2 in the binding protein are heavy chainvariable domains. In another embodiment, the heavy chain variable domainis selected from the group consisting of a murine heavy chain variabledomain, a human heavy chain variable domain, a CDR grafted heavy chainvariable domain, and a humanized heavy chain variable domain. In yetanother, embodiment VD1 and VD2 bind the same antigen. In anotherembodiment VD1 and VD2 bind different antigens. In still anotherembodiment, C is a heavy chain constant domain. For example, X1 is alinker with the proviso that X1 is not CH1. For example, X1 is a linkerselected from the group consisting of AKTTPKLEEGEFSEAR (SEQ ID NO: 1);AKTTPKLEEGEFSEARV (SEQ ID NO: 2); AKTTPKLGG (SEQ ID NO: 3); SAKTTPKLGG(SEQ ID NO: 4); SAKTTP (SEQ ID NO: 5); RADAAP (SEQ ID NO: 6); RADAAPTVS(SEQ ID NO: 7); RADAAAAGGPGS (SEQ ID NO: 8); RADAAAA(G₄S)₄ (SEQ ID NO:9); SAKTTPKLEEGEFSEARV (SEQ ID NO: 10); ADAAP (SEQ ID NO: 11);ADAAPTVSIFPP (SEQ ID NO: 12); TVAAP (SEQ ID NO: 13); TVAAPSVFIFPP (SEQID NO: 14); QPKAAP (SEQ ID NO: 15); QPKAAPSVTLFPP (SEQ ID NO: 16);AKTTPP (SEQ ID NO: 17); AKTTPPSVTPLAP (SEQ ID NO: 18); AKTTAP (SEQ IDNO: 19); AKTTAPSVYPLAP (SEQ ID NO: 20); ASTKGP (SEQ ID NO: 21);ASTKGPSVFPLAP (SEQ ID NO: 22), GGGGSGGGGSGGGGS (SEQ ID NO: 23);GENKVEYAPALMALS (SEQ ID NO: 24); GPAKELTPLKEAKVS (SEQ ID NO: 25);GHEAAAVMQVQYPAS (SEQ ID NO: 26); TVAAPSVFIFPPTVAAPSVFIFPP (SEQ ID NO:27); and ASTKGPSVFPLAPASTKGPSVFPLAP (SEQ ID NO: 28). In an embodiment,X2 is an Fc region. In another embodiment, X2 is a variant Fc region.

In an embodiment, the binding proteins disclosed herein comprises apolypeptide chain, wherein the polypeptide chain comprisesVD1-(X1)n-VD2-C-(X2)n, wherein VD1 is a first heavy chain variabledomain, VD2 is a second heavy chain variable domain, C is a heavy chainconstant domain, X1 is a linker with the proviso that it is not CH1, andX2 is an Fc region.

In an embodiment, VD1 and VD2 in the binding protein are light chainvariable domains. In an embodiment, the light chain variable domain isselected from the group consisting of a murine light chain variabledomain, a human light chain variable domain, a CDR grafted light chainvariable domain, and a humanized light chain variable domain. In oneembodiment VD1 and VD2 bind the same antigen. In another embodiment VD1and VD2 bind different antigens. In an embodiment, C is a light chainconstant domain. In another embodiment, X1 is a linker with the provisothat X1 is not CL1. In an embodiment, X1 is a linker selected from thegroup consisting of AKTTPKLEEGEFSEAR (SEQ ID NO: 1); AKTTPKLEEGEFSEARV(SEQ ID NO: 2); AKTTPKLGG (SEQ ID NO: 3); SAKTTPKLGG (SEQ ID NO: 4);SAKTTP (SEQ ID NO: 5); RADAAP (SEQ ID NO: 6); RADAAPTVS (SEQ ID NO: 7);RADAAAAGGPGS (SEQ ID NO: 8); RADAAAA(G₄S)₄ (SEQ ID NO: 9);SAKTTPKLEEGEFSEARV (SEQ ID NO: 10); ADAAP (SEQ ID NO: 11); ADAAPTVSIFPP(SEQ ID NO: 12); TVAAP (SEQ ID NO: 13); TVAAPSVFIFPP (SEQ ID NO: 14);QPKAAP (SEQ ID NO: 15); QPKAAPSVTLFPP (SEQ ID NO: 16); AKTTPP (SEQ IDNO: 17); AKTTPPSVTPLAP (SEQ ID NO: 18); AKTTAP (SEQ ID NO: 19);AKTTAPSVYPLAP (SEQ ID NO: 20); ASTKGP (SEQ ID NO: 21); ASTKGPSVFPLAP(SEQ ID NO: 22), GGGGSGGGGSGGGGS (SEQ ID NO: 23); GENKVEYAPALMALS (SEQID NO: 24); GPAKELTPLKEAKVS (SEQ ID NO: 25); GHEAAAVMQVQYPAS (SEQ ID NO:26); TVAAPSVFIFPPTVAAPSVFIFPP (SEQ ID NO: 27); andASTKGPSVFPLAPASTKGPSVFPLAP (SEQ ID NO: 28). In an embodiment, thebinding protein does not comprise X2.

In an embodiment, both the variable heavy and variable light chainscomprise the same linker In another embodiment, the variable heavy andvariable light chains comprise different linkers. In another embodiment,both the variable heavy and variable light chains comprise a short(about 6 amino acids) linker. In another embodiment, both the variableheavy and variable light chains comprise a long (greater than 6 aminoacids) linker. In another embodiment, the variable heavy chain comprisesa short linker and the variable light chain comprises a long linker. Inanother embodiment, the variable heavy chain comprises a long linker andthe variable light chain comprises a short linker.

In an embodiment, the binding proteins disclosed herein comprises apolypeptide chain, wherein said polypeptide chain comprisesVD1-(X1)n-VD2-C-(X2)n, wherein VD1 is a first light chain variabledomain, VD2 is a second light chain variable domain, C is a light chainconstant domain, X1 is a linker with the proviso that it is not CH1, andX2 does not comprise an Fc region.

In another embodiment the invention provides a binding proteincomprising two polypeptide chains, wherein said first polypeptide chaincomprises VD1-(X1)n-VD2-C-(X2)n, wherein VD1 is a first heavy chainvariable domain, VD2 is a second heavy chain variable domain, C is aheavy chain constant domain, X1 is a linker with the proviso that it isnot CH1, and X2 is an Fc region; and said second polypeptide chaincomprises VD1-(X1)n-VD2-C-(X2)n, wherein VD1 is a first light chainvariable domain, VD2 is a second light chain variable domain, C is alight chain constant domain, X1 is a linker with the proviso that it isnot CH1, and X2 does not comprise an Fc region. In a particularembodiment, the Dual Variable Domain (DVD) binding protein comprisesfour polypeptide chains wherein the first two polypeptide chainscomprises VD1-(X1)n-VD2-C-(X2)n, respectively wherein VD1 is a firstheavy chain variable domain, VD2 is a second heavy chain variabledomain, C is a heavy chain constant domain, X1 is a linker with theproviso that it is not CH1, and X2 is an Fc region; and the second twopolypeptide chain comprises VD1-(X1)n-VD2-C-(X2)n respectively, whereinVD1 is a first light chain variable domain, VD2 is a second light chainvariable domain, C is a light chain constant domain, X1 is a linker withthe proviso that it is not CH1, and X2 does not comprise an Fc region.Such a Dual Variable Domain (DVD) binding protein has four antigenbinding sites.

In another embodiment, the binding proteins disclosed herein bind one ormore targets. In an embodiment, the target is selected from the groupconsisting of cytokines, cell surface proteins, enzymes and receptors.In another embodiment, the binding protein modulates a biologicalfunction of one or more targets. In another embodiment, the bindingprotein neutralizes one or more targets. The binding proteins of theinvention bind cytokines selected from the group consisting oflymphokines, monokines, polypeptide hormones, receptors, or tumormarkers. For example, the DVD-Igs of the invention are capable ofbinding two or more of the following: Interleukin-1 alpha (IL-1α) andInterleukin-1 beta (IL-1β).

In an embodiment, the binding protein that binds IL-1α (seq. 3) andIL-1β (seq. 1) comprises a DVD-Ig heavy chain amino acid sequence of SEQID NO. 52 and a DVD-Ig light chain amino acid sequence of SEQ ID NO. 53.

In an embodiment, the binding protein that binds IL-1α (seq. 2) andIL-1β (seq. 1) comprises a DVD-Ig heavy chain amino acid sequenceselected from the group consisting of SEQ ID NO. 54 and SEQ ID NO. 56;and a DVD-Ig light chain amino acid sequence selected from the groupconsisting of SEQ ID NO. 55 and SEQ ID NO. 57. In an embodiment, thebinding protein that binds IL-1α (seq. 2) and IL-1β (seq. 1) comprises aDVD-Ig heavy chain amino acid sequence of SEQ ID NO. 54 and a DVD-Iglight chain amino acid sequence of SEQ ID NO: 55. In another embodiment,the binding protein that binds IL-1α (seq. 2) and IL-1β (seq. 1)comprises a DVD-Ig heavy chain amino acid sequence of SEQ ID NO. 56 anda DVD-Ig light chain amino acid sequence of SEQ ID NO: 57.

In an embodiment, the binding protein that binds IL-1α (seq. 1) andIL-1β (seq. 1) comprises a DVD-Ig heavy chain amino acid sequenceselected from the group consisting of SEQ ID NO. 58 and SEQ ID NO. 60;and a DVD-Ig light chain amino acid sequence selected from the groupconsisting of SEQ ID NO. 59 and SEQ ID NO. 61. In an embodiment, thebinding protein that binds IL-1α (seq. 1) and IL-1β (seq. 1) comprises aDVD-Ig heavy chain amino acid sequence of SEQ ID NO. 58 and a DVD-Iglight chain amino acid sequence of SEQ ID NO: 59. In another embodiment,the binding protein that binds IL-1α (seq. 1) and IL-1β (seq. 1)comprises a DVD-Ig heavy chain amino acid sequence of SEQ ID NO. 60 anda DVD-Ig light chain amino acid sequence of SEQ ID NO: 61.

In an embodiment, the binding protein that binds IL-1α (seq. 3) andIL-1β (seq. 2) comprises a DVD-Ig heavy chain amino acid sequence of SEQID NO. 62 and a DVD-Ig light chain amino acid sequence of SEQ ID NO. 63.

In an embodiment, the binding protein that binds IL-1α (seq. 4) andIL-1β (seq. 2) comprises a DVD-Ig heavy chain amino acid sequence of SEQID NO. 64 and a DVD-Ig light chain amino acid sequence of SEQ ID NO. 65.

In an embodiment, the binding protein that binds IL-1α (seq. 4) andIL-1β (seq. 3) comprises a DVD-Ig heavy chain amino acid sequence of SEQID NO. 66 and a DVD-Ig light chain amino acid sequence of SEQ ID NO. 67.

In an embodiment, the binding protein that binds IL-1α (seq. 4) andIL-1β (seq. 4) comprises a DVD-Ig heavy chain amino acid sequence of SEQID NO. 68 and a DVD-Ig light chain amino acid sequence of SEQ ID NO. 69.

In an embodiment, the binding protein that binds IL-1α (seq. 4) andIL-1β (seq. 5) comprises a DVD-Ig heavy chain amino acid sequence of SEQID NO. 70 and a DVD-Ig light chain amino acid sequence of SEQ ID NO. 71.

In another embodiment the invention provides a binding proteincomprising a polypeptide chain, wherein said polypeptide chain comprisesVD1-(X1)n-VD2-C-(X2)n, wherein; VD1 is a first heavy chain variabledomain obtained from a first parent antibody, or antigen binding portionthereof; VD2 is a second heavy chain variable domain obtained from asecond parent antibody, or antigen binding portion thereof, which can bethe same or different from the first parent antibody; C is a heavy chainconstant domain; (X1)n is a linker with the proviso that it is not CH1,wherein said (X1)n is either present or absent; and (X2)n is an Fcregion, wherein said (X2)n is either present or absent. In anembodiment, the Fc region is absent from the binding protein.

In another embodiment, the invention provides a binding proteincomprising a polypeptide chain, wherein said polypeptide chain comprisesVD1-(X1)n-VD2-C-(X2)n, wherein, VD1 is a first light chain variabledomain obtained from a first parent antibody or antigen binding portionthereof; VD2 is a second light chain variable domain obtained from asecond parent antibody or antigen binding portion thereof, which can bethe same or different from the first parent antibody; C is a light chainconstant domain; (X1)n is a linker with the proviso that it is not CH1,wherein said (X1)n is either present or absent; and (X2)n does notcomprise an Fc region, wherein said (X2)n is either present or absent.In an embodiment, (X2)n is absent from the binding protein.

In another embodiment the binding protein of the invention comprisesfirst and second polypeptide chains, wherein said first polypeptidechain comprises a first VD1-(X1)n-VD2-C-(X2)n, wherein VD1 is a firstheavy chain variable domain obtained from a first parent antibody orantigen binding portion thereof; VD2 is a second heavy chain variabledomain obtained from a second parent antibody or antigen binding portionthereof, which can be the same or different from the first parentantibody; C is a heavy chain constant domain; (X1)n is a linker with theproviso that it is not CH1, wherein said (X1)n is either present orabsent; and (X2)n is an Fc region, wherein said (X2)n is either presentor absent; and wherein said second polypeptide chain comprises a secondVD1-(X1)n-VD2-C-(X2)n, wherein VD1 is a first light chain variabledomain obtained from a first parent antibody or antigen binding portionthereof; VD2 is a second light chain variable domain obtained from asecond parent antibody or antigen binding portion thereof, which can bethe same or different from the first parent antibody; C is a light chainconstant domain; (X1)n is a linker with the proviso that it is not CH1,wherein said (X1)n is either present or absent; and (X2)n does notcomprise an Fc region, wherein said (X2)n is either present or absent.In another embodiment, the binding protein comprises two firstpolypeptide chains and two second polypeptide chains. In yet anotherembodiment, (X2)n is absent from the second polypeptide. In stillanother embodiment, the Fc region, if present in the first polypeptideis selected from the group consisting of native sequence Fc region and avariant sequence Fc region. In still another embodiment, the Fc regionis selected from the group consisting of an Fc region from an IgG1,IgG2, IgG3, IgG4, IgA, IgM, IgE, and an IgD.

In another embodiment the binding protein of the invention is a DVD-Igthat binds two antigens comprising four polypeptide chains, wherein,each of the first and third polypeptide chains compriseVD1-(X1)n-VD2-C-(X2)n, wherein, VD1 is a first heavy chain variabledomain obtained from a first parent antibody or antigen binding portionthereof; VD2 is a second heavy chain variable domain obtained from asecond parent antibody or antigen binding portion thereof, which can bethe same as or different from the first parent antibody; C is a heavychain constant domain; (X1)n is a linker with the proviso that it is notCH1, wherein said (X1)n is either present or absent; and (X2)n is an Fcregion, wherein said (X2)n is either present or absent; and wherein eachof the second and fourth polypeptide chains compriseVD1-(X1)n-VD2-C-(X2)n, wherein VD1 is a first light chain variabledomain obtained from a first parent antibody or antigen binding portionthereof; VD2 is a second light chain variable domain obtained from asecond parent antibody or antigen binding portion thereof, which can bethe same as or different from the first parent antibody; C is a lightchain constant domain; (X1)n is a linker with the proviso that it is notCH1, wherein said (X1)n is either present or absent; and (X2)n does notcomprise an Fc region, wherein said (X2)n is either present or absent.

The invention provides a method of making a DVD-Ig binding protein bypreselecting the parent antibodies. In an embodiment, the method ofmaking a Dual Variable Domain Immunoglobulin that binds two antigenscomprising the steps of a) obtaining a first parent antibody or antigenbinding portion thereof, that binds a first antigen; b) obtaining asecond parent antibody or antigen binding portion thereof, that binds asecond antigen; c) constructing first and third polypeptide chains, eachof which comprises VD1-(X1)n-VD2-C-(X2)n, wherein, VD1 is a first heavychain variable domain obtained from said first parent antibody orantigen binding portion thereof; VD2 is a second heavy chain variabledomain obtained from said second parent antibody or antigen bindingportion thereof, which can be the same as or different from the firstparent antibody; C is a heavy chain constant domain; (X1)n is a linkerwith the proviso that it is not CH1, wherein said (X1)n is eitherpresent or absent; and (X2)n is an Fc region, wherein said (X2)n iseither present or absent; d) constructing second and fourth polypeptidechains, each of which comprises VD1-(X1)n-VD2-C-(X2)n, wherein, VD1 is afirst light chain variable domain obtained from said first parentantibody or antigen binding portion thereof; VD2 is a second light chainvariable domain obtained from said second parent antibody or antigenbinding thereof, which can be the same as or different from the firstparent antibody; C is a light chain constant domain; (X1)n is a linkerwith the proviso that it is not CH1, wherein said (X1)n is eitherpresent or absent; and (X2)n does not comprise an Fc region, whereinsaid (X2)n is either present or absent; and e) expressing said first,second, third and fourth polypeptide chains; such that a DVD-Ig moleculethat binds said first and said second antigen is generated.

In still another embodiment, the invention provides a method ofgenerating a DVD-Ig molecule that binds two antigens with desiredproperties comprising the steps of a) obtaining a first parent antibodyor antigen binding portion thereof, that binds a first antigen andpossessing at least one desired property exhibited by the DVD-Igmolecule; b) obtaining a second parent antibody or antigen bindingportion thereof, that binds a second antigen and possessing at least onedesired property exhibited by the DVD-Ig molecule; c) constructing firstand third polypeptide chains comprising VD1-(X1)n-VD2-C-(X2)n, wherein;VD1 is a first heavy chain variable domain obtained from said firstparent antibody or antigen binding portion thereof; VD2 is a secondheavy chain variable domain obtained from said second parent antibody orantigen binding portion thereof, which can be the same as or differentfrom the first parent antibody; C is a heavy chain constant domain;(X1)n is a linker with the proviso that it is not CH1, wherein said(X1)n is either present or absent; and (X2)n is an Fc region, whereinsaid (X2)n is either present or absent; d) constructing second andfourth polypeptide chains comprising VD1-(X1)n-VD2-C-(X2)n, wherein; VD1is a first light chain variable domain obtained from said first parentantibody or antigen binding portion thereof; VD2 is a second light chainvariable domain obtained from said second parent antibody or antigenbinding portion thereof, which can be the same as or different from thefirst parent antibody; C is a light chain constant domain; (X1)n is alinker with the proviso that it is not CH1, wherein said (X1)n is eitherpresent or absent; and (X2)n does not comprise an Fc region, whereinsaid (X2)n is either present or absent; e) expressing said first,second, third and fourth polypeptide chains; such that a Dual VariableDomain Immunoglobulin that binds said first and said second antigen withdesired properties is generated.

In one embodiment, the VD1of the first and second polypeptide chainsdisclosed herein are obtained from the same parent antibody or antigenbinding portion thereof. In another embodiment, the VD1of the first andsecond polypeptide chains disclosed herein are obtained from differentparent antibodies or antigen binding portions thereof. In anotherembodiment, the VD2 of the first and second polypeptide chains disclosedherein are obtained from the same parent antibody or antigen bindingportion thereof. In another embodiment, the VD2 of the first and secondpolypeptide chains disclosed herein are obtained from different parentantibodies or antigen binding portions thereof.

In one embodiment the first parent antibody or antigen binding portionthereof, and the second parent antibody or antigen binding portionthereof, are the same antibody. In another embodiment the first parentantibody or antigen binding portion thereof, and the second parentantibody or antigen binding portion thereof, are different antibodies.

In one embodiment the first parent antibody or antigen binding portionthereof, binds a first antigen and the second parent antibody or antigenbinding portion thereof, binds a second antigen. In a particularembodiment, the first and second antigens are the same antigen. Inanother embodiment, the parent antibodies bind different epitopes on thesame antigen. In another embodiment the first and second antigens aredifferent antigens. In another embodiment, the first parent antibody orantigen binding portion thereof, binds the first antigen with a potencydifferent from the potency with which the second parent antibody orantigen binding portion thereof, binds the second antigen. In yetanother embodiment, the first parent antibody or antigen binding portionthereof, binds the first antigen with an affinity different from theaffinity with which the second parent antibody or antigen bindingportion thereof, binds the second antigen.

In another embodiment the first parent antibody or antigen bindingportion thereof, and the second parent antibody or antigen bindingportion thereof, are selected from the group consisting of, humanantibody, CDR grafted antibody, and humanized antibody. In anembodiment, the antigen binding portions are selected from the groupconsisting of a Fab fragment, a F(ab′)₂ fragment, a bivalent fragmentcomprising two Fab fragments linked by a disulfide bridge at the hingeregion; a Fd fragment consisting of the VH and CH1 domains; a Fvfragment consisting of the VL and VH domains of a single arm of anantibody, a dAb fragment, an isolated complementarity determining region(CDR), a single chain antibody, and diabodies.

In another embodiment the binding protein of the invention possesses atleast one desired property exhibited by the first parent antibody orantigen binding portion thereof, or the second parent antibody orantigen binding portion thereof. Alternatively, the first parentantibody or antigen binding portion thereof and the second parentantibody or antigen binding portion thereof possess at least one desiredproperty exhibited by the Dual Variable Domain Immunoglobulin. In anembodiment, the desired property is selected from one or more antibodyparameters. In another embodiment, the antibody parameters are selectedfrom the group consisting of antigen specificity, affinity to antigen,potency, biological function, epitope recognition, stability,solubility, production efficiency, immunogenicity, pharmacokinetics,bioavailability, tissue cross reactivity, and orthologous antigenbinding. In an embodiment the binding protein is multivalent. In anotherembodiment, the binding protein is multispecific. The multivalent and ormultispecific binding proteins described herein have desirableproperties particularly from a therapeutic standpoint. For instance, themultivalent and or multispecific binding protein may (1) be internalized(and/or catabolized) faster than a bivalent antibody by a cellexpressing an antigen to which the antibodies bind; (2) be an agonistantibody; and/or (3) induce cell death and/or apoptosis of a cellexpressing an antigen to which the multivalent antibody binds to. The“parent antibody” which provides at least one antigen bindingspecificity of the multivalent and or multispecific binding proteins maybe one which is internalized (and/or catabolized) by a cell expressingan antigen to which the antibody binds; and/or may be an agonist, celldeath-inducing, and/or apoptosis-inducing antibody, and the multivalentand or multispecific binding protein as described herein may displayimprovement(s) in one or more of these properties. Moreover, the parentantibody may lack any one or more of these properties, but may beendowed with them when constructed as a multivalent binding protein asdescribed herein.

In another embodiment the binding protein of the invention has an onrate constant (Kon) to one or more targets selected from the groupconsisting of: at least about 10²M⁻¹s⁻¹; at least about 10³M⁻¹s⁻¹; atleast about 10⁴M⁻¹s⁻¹; at least about 10⁵M⁻¹s⁻¹; and at least about10⁶M⁻¹s⁻¹, as measured by surface plasmon resonance. In an embodiment,the binding protein of the invention has an on rate constant (Kon) toone or more targets between about 10²M⁻¹s⁻¹ and about 10³M⁻¹s⁻¹; betweenabout 10³M⁻¹s⁻¹ and about 10⁴M⁻¹s⁻¹; between about 10⁴M⁻¹s⁻¹ and about10⁵M⁻¹s⁻¹; or between about 10⁵M⁻¹s⁻¹ and about 10⁶M⁻¹s⁻¹, as measuredby surface plasmon resonance.

In another embodiment the binding protein has an off rate constant(Koff) for one or more targets selected from the group consisting of: atmost about 10⁻³s⁻¹; at most about 10⁻⁴s⁻¹; at most about 10⁻⁵s⁻¹; and atmost about 10⁻⁶s⁻¹, as measured by surface plasmon resonance. In anembodiment, the binding protein of the invention has an off rateconstant (Koff) to one or more targets of about 10⁻³s⁻¹ to about10⁻⁴s⁻¹; of about 10⁻⁴s⁻¹ to about 10⁻⁵s⁻¹; or of about 10⁻⁵s⁻¹ to about10⁻⁶s⁻¹, as measured by surface plasmon resonance.

In another embodiment the binding protein has a dissociation constant(K_(D)) to one or more targets selected from the group consisting of: atmost about 10⁻⁷ M; at most about 10⁻⁸ M; at most about 10⁻⁹ M; at mostabout 10⁻¹⁰ M; at most about 10⁻¹¹ M; at most about 10⁻¹² M; and at mostabout 10⁻¹³M. In an embodiment, the binding protein of the invention hasa dissociation constant (K_(D)) to its targets of from about 10⁻⁷ M toabout 10⁻⁸ M; of from about 10⁻⁸ M to about 10⁻⁹ M; of from about 10⁻⁹ Mto about 10⁻¹⁰ M; of from about 10⁻¹⁰ to about 10⁻¹¹ M; of from about10⁻¹¹ M to about 10⁻¹² M; or of from about 10⁻¹² M to about 10⁻¹³ M.

In another embodiment, the binding proteins described herein areconjugates further comprising an agent selected from the groupconsisting of an immunoadhesion molecule, an imaging agent, atherapeutic agent, and a cytotoxic agent. In an embodiment, the imagingagent is selected from the group consisting of a radiolabel, an enzyme,a fluorescent label, a luminescent label, a bioluminescent label, amagnetic label, and biotin. In another embodiment, the imaging agent isa radiolabel selected from the group consisting of: ³H, ¹⁴C, ³⁵S, ⁹⁰Y,⁹⁹Tc, ¹¹¹In, ¹²⁵I, ¹³¹I, ¹⁷⁷Lu, ¹⁶⁶Ho, and ¹⁵³Sm. In yet anotherembodiment, the therapeutic or cytotoxic agent is selected from thegroup consisting of an anti-metabolite, an alkylating agent, anantibiotic, a growth factor, a cytokine, an anti-angiogenic agent, ananti-mitotic agent, an anthracycline, toxin, and an apoptotic agent.

In another embodiment, the binding proteins described herein binds to acellular protein and an agent selected from the group consisting of animmunoadhesion molecule, an imaging agent, a therapeutic agent, and acytotoxic agent. In an embodiment, the imaging agent is selected fromthe group consisting of a radiolabel, an enzyme, a fluorescent label, aluminescent label, a bioluminescent label, a magnetic label, and biotin.In another embodiment, the imaging agent is a radiolabel selected fromthe group consisting of ³H, ¹⁴C, ³⁵S, ⁹⁰Y, ⁹⁹Tc, ¹¹¹In, ¹²⁵I, ¹³¹I,¹⁷⁷Lu, ¹⁶⁶Ho, and ^(sure)Sm. In yet another embodiment, the therapeuticor cytotoxic agent is selected from the group consisting of ananti-metabolite, an alkylating agent, an antibiotic, a growth factor, acytokine, an anti-angiogenic agent, an anti-mitotic agent, ananthracycline, toxin, and an apoptotic agent.

In another embodiment, the binding proteins described herein are acrystallized binding protein and exists as a crystal. In an embodiment,the crystal is a carrier-free pharmaceutical controlled release crystal.In yet another embodiment, the crystallized binding protein has agreater half life in vivo than the soluble counterpart of said bindingprotein. In still another embodiment, the crystallized binding proteinretains biological activity.

In another embodiment, the binding proteins described herein areglycosylated. For example, the glycosylation is a human glycosylationpattern.

One aspect of the invention pertains to an isolated nucleic acidencoding any one of the binding proteins disclosed herein. A furtherembodiment provides a vector comprising the isolated nucleic aciddisclosed herein wherein said vector is selected from the groupconsisting of pcDNA; pTT (Durocher et al. (2002) Nucl. Acids Res. 30:2;pTT3 (pTT with additional multiple cloning site; pEFBOS (Mizushima andNagata, (1990) Nucl. Acids Res. 18:17); pBV; NV; pcDNA3.1 TOPO, pEF6TOPO and pBJ. In an embodiment, the vector is a vector disclosed in USPatent Publication No. 20090239259.

In another aspect a host cell is transformed with the vector disclosedherein. In an embodiment, the host cell is a prokaryotic cell. Inanother embodiment, the host cell is E. Coli. In a related embodimentthe host cell is a eukaryotic cell. In another embodiment, theeukaryotic cell is selected from the group consisting of a protist cell,an animal cell, a plant cell and a fungal cell. In yet anotherembodiment, the host cell is a mammalian cell including, but not limitedto, CHO, COS; NS0, SP2, PER.C6 or a fungal cell such as Saccharomycescerevisiae; or an insect cell such as Sf9.

In an embodiment, two or more DVD-Igs, e.g., with differentspecificities, are produced in a single recombinant host cell. Forexample, the expression of a mixture of antibodies has been calledOligoclonics™ Merus B. V., The Netherlands; U.S. Pat. Nos. 7,262,028 and7,429,486.

Another aspect of the invention provides a method of producing a bindingprotein disclosed herein comprising culturing any one of the host cellsalso disclosed herein in a culture medium under conditions sufficient toproduce the binding protein. In an embodiment, 50%-75% of the bindingprotein produced by this method is a dual specific tetravalent bindingprotein. In a particular embodiment, 75%-90% of the binding proteinproduced by this method is a dual specific tetravalent binding protein.In a particular embodiment, 90%-95% of the binding protein produced is adual specific tetravalent binding protein.

One embodiment provides a composition for the release of a bindingprotein wherein the composition comprises a formulation that in turncomprises a crystallized binding protein, as disclosed herein, and aningredient, and at least one polymeric carrier. For example, thepolymeric carrier comprises one or more polymers selected from the groupconsisting of poly (acrylic acid), poly (cyanoacrylates), poly (aminoacids), poly (anhydrides), poly (depsipeptide), poly (esters), poly(lactic acid), poly (lactic-co-glycolic acid) or PLGA, poly(b-hydroxybutryate), poly (caprolactone), poly (dioxanone); poly(ethylene glycol), poly ((hydroxypropyl)methacrylamide,poly[(organo)phosphazene], poly (ortho esters), poly (vinyl alcohol),poly (vinylpyrrolidone), maleic anhydride-alkyl vinyl ether copolymers,pluronic polyols, albumin, alginate, cellulose and cellulosederivatives, collagen, fibrin, gelatin, hyaluronic acid,oligosaccharides, glycaminoglycans, sulfated polysaccharides, blends andcopolymers thereof. For example, the ingredient is selected from thegroup consisting of albumin, sucrose, trehalose, lactitol, gelatin,hydroxypropyl-β-cyclodextrin, methoxypolyethylene glycol andpolyethylene glycol. Another embodiment provides a method for treating amammal comprising the step of administering to the mammal an effectiveamount of the composition disclosed herein.

The invention also provides a pharmaceutical composition comprising abinding protein, as disclosed herein and a pharmaceutically acceptablecarrier. In a further embodiment the pharmaceutical compositioncomprises at least one additional therapeutic agent for treating adisorder. For example, the additional agent is selected from the groupconsisting of: a therapeutic agent, an imaging agent, a cytotoxic agent,an angiogenesis inhibitor (including but not limited to an anti-VEGFantibody or a VEGF-trap), a kinase inhibitor (including but not limitedto a KDR and a TIE-2 inhibitor), a co-stimulation molecule blocker(including but not limited to anti-B7.1, anti-B7.2, CTLA4-Ig,anti-CD20), an adhesion molecule blocker (including but not limited toan anti-LFA-1 antibody, an anti-E/L selectin antibody, a small moleculeinhibitor), an anti-cytokine antibody or functional fragment thereof(including but not limited to an anti-IL-18, an anti-TNF, and ananti-IL-6/cytokine receptor antibody), methotrexate, cyclosporin,rapamycin, FK506, a detectable label or reporter, a TNF antagonist, anantirheumatic, a muscle relaxant, a narcotic, a non-steroidanti-inflammatory drug (NSAID), an analgesic, an anesthetic, a sedative,a local anesthetic, a neuromuscular blocker, an antimicrobial, anantipsoriatic, a corticosteriod, an anabolic steroid, an erythropoietin,an immunization, an immunoglobulin, an immunosuppressive, a growthhormone, a hormone replacement drug, a radiopharmaceutical, anantidepressant, an antipsychotic, a stimulant, an asthma medication, abeta agonist, an inhaled steroid, an epinephrine or analog, a cytokine,and a cytokine antagonist.

In another aspect, the invention provides a method for treating a humansubject suffering from a disorder in which the target, or targets,capable of being bound by the binding protein disclosed herein isdetrimental, comprising administering to the human subject a bindingprotein disclosed herein such that the activity of the target, ortargets in the human subject is inhibited and one of more symptoms isalleviated or treatment is achieved. For example, the disorder isselected from the group comprising arthritis, osteoarthritis, juvenilechronic arthritis, septic arthritis, Lyme arthritis, psoriaticarthritis, reactive arthritis, spondyloarthropathy, systemic lupuserythematosus, Crohn's disease, ulcerative colitis, inflammatory boweldisease, insulin dependent diabetes mellitus, thyroiditis, asthma,allergic diseases, psoriasis, dermatitis scleroderma, graft versus hostdisease, organ transplant rejection, acute or chronic immune diseaseassociated with organ transplantation, sarcoidosis, atherosclerosis,disseminated intravascular coagulation, Kawasaki's disease, Grave'sdisease, nephrotic syndrome, chronic fatigue syndrome, Wegener'sgranulomatosis, Henoch-Schoenlein purpurea, microscopic vasculitis ofthe kidneys, chronic active hepatitis, uveitis, septic shock, toxicshock syndrome, sepsis syndrome, cachexia, infectious diseases,parasitic diseases, acquired immunodeficiency syndrome, acute transversemyelitis, Huntington's chorea, Parkinson's disease, Alzheimer's disease,stroke, primary biliary cirrhosis, hemolytic anemia, malignancies, heartfailure, myocardial infarction, Addison's disease, sporadicpolyglandular deficiency type I and polyglandular deficiency type II,Schmidt's syndrome, adult (acute) respiratory distress syndrome,alopecia, alopecia greata, seronegative arthopathy, arthropathy,Reiter's disease, psoriatic arthropathy, ulcerative colitic arthropathy,enteropathic synovitis, chlamydia, yersinia and salmonella associatedarthropathy, spondyloarthopathy, atheromatous disease/arteriosclerosis,atopic allergy, autoimmune bullous disease, pemphigus vulgaris,pemphigus foliaceus, pemphigoid, linear IgA disease, autoimmunehaemolytic anaemia, Coombs positive haemolytic anaemia, acquiredpernicious anaemia, juvenile pernicious anaemia, myalgicencephalitis/Royal Free Disease, chronic mucocutaneous candidiasis,giant cell arteritis, primary sclerosing hepatitis, cryptogenicautoimmune hepatitis, Acquired Immunodeficiency Disease Syndrome,Acquired Immunodeficiency Related Diseases, Hepatitis B, Hepatitis C,common varied immunodeficiency (common variable hypogammaglobulinaemia),dilated cardiomyopathy, female infertility, ovarian failure, prematureovarian failure, fibrotic lung disease, cryptogenic fibrosingalveolitis, post-inflammatory interstitial lung disease, interstitialpneumonitis, connective tissue disease associated interstitial lungdisease, mixed connective tissue disease associated lung disease,systemic sclerosis associated interstitial lung disease, rheumatoidarthritis associated interstitial lung disease, systemic lupuserythematosus associated lung disease, dermatomyositis/polymyositisassociated lung disease, Sjögren's disease associated lung disease,ankylosing spondylitis associated lung disease, vasculitic diffuse lungdisease, haemosiderosis associated lung disease, drug-inducedinterstitial lung disease, fibrosis, radiation fibrosis, bronchiolitisobliterans, chronic eosinophilic pneumonia, lymphocytic infiltrativelung disease, postinfectious interstitial lung disease, gouty arthritis,autoimmune hepatitis, type-1 autoimmune hepatitis (classical autoimmuneor lupoid hepatitis), type-2 autoimmune hepatitis (anti-LKM antibodyhepatitis), autoimmune mediated hypoglycemia, type B insulin resistancewith acanthosis nigricans, hypoparathyroidism, acute immune diseaseassociated with organ transplantation, chronic immune disease associatedwith organ transplantation, osteoarthrosis, primary sclerosingcholangitis, psoriasis type 1, psoriasis type 2, idiopathic leucopaenia,autoimmune neutropaenia, renal disease NOS, glomerulonephritides,microscopic vasulitis of the kidneys, lyme disease, discoid lupuserythematosus, male infertility idiopathic or NOS, sperm autoimmunity,multiple sclerosis (all subtypes), sympathetic ophthalmia, pulmonaryhypertension secondary to connective tissue disease, Goodpasture'ssyndrome, pulmonary manifestation of polyarteritis nodosa, acuterheumatic fever, rheumatoid spondylitis, Still's disease, systemicsclerosis, Sjörgren's syndrome, Takayasu's disease/arteritis, autoimmunethrombocytopaenia, idiopathic thrombocytopaenia, autoimmune thyroiddisease, hyperthyroidism, goitrous autoimmune hypothyroidism(Hashimoto's disease), atrophic autoimmune hypothyroidism, primarymyxoedema, phacogenic uveitis, primary vasculitis, vitiligo acute liverdisease, chronic liver diseases, alcoholic cirrhosis, alcohol-inducedliver injury, choleosatatis, idiosyncratic liver disease, Drug-Inducedhepatitis, Non-alcoholic Steatohepatitis, allergy and asthma, group Bstreptococci (GBS) infection, mental disorders (e.g., depression andschizophrenia), Th2 Type and Th1 Type mediated diseases, acute andchronic pain (different forms of pain), and cancers such as lung,breast, stomach, bladder, colon, pancreas, ovarian, prostate and rectalcancer and hematopoietic malignancies (leukemia and lymphoma),Abetalipoprotemia, Acrocyanosis, acute and chronic parasitic orinfectious processes, acute leukemia, acute lymphoblastic leukemia(ALL), acute myeloid leukemia (AML), acute or chronic bacterialinfection, acute pancreatitis, acute renal failure, adenocarcinomas,aerial ectopic beats, AIDS dementia complex, alcohol-induced hepatitis,allergic conjunctivitis, allergic contact dermatitis, allergic rhinitis,allograft rejection, alpha-1-antitrypsin deficiency, amyotrophic lateralsclerosis, anemia, angina pectoris, anterior horn cell degeneration,anti cd3 therapy, antiphospholipid syndrome, anti-receptorhypersensitivity reactions, aortic and peripheral aneuryisms, aorticdissection, arterial hypertension, arteriosclerosis, arteriovenousfistula, ataxia, atrial fibrillation (sustained or paroxysmal), atrialflutter, atrioventricular block, B cell lymphoma, bone graft rejection,bone marrow transplant (BMT) rejection, bundle branch block, Burkitt'slymphoma, Burns, cardiac arrhythmias, cardiac stun syndrome, cardiactumors, cardiomyopathy, cardiopulmonary bypass inflammation response,cartilage transplant rejection, cerebellar cortical degenerations,cerebellar disorders, chaotic or multifocal atrial tachycardia,chemotherapy associated disorders, chronic myelocytic leukemia (CML),chronic alcoholism, chronic inflammatory pathologies, chroniclymphocytic leukemia (CLL), chronic obstructive pulmonary disease(COPD), chronic salicylate intoxication, colorectal carcinoma,congestive heart failure, conjunctivitis, contact dermatitis, corpulmonale, coronary artery disease, Creutzfeldt-Jakob disease, culturenegative sepsis, cystic fibrosis, cytokine therapy associated disorders,Dementia pugilistica, demyelinating diseases, dengue hemorrhagic fever,dermatitis, dermatologic conditions, diabetes, diabetes mellitus,diabetic ateriosclerotic disease, Diffuse Lewy body disease, dilatedcongestive cardiomyopathy, disorders of the basal ganglia, Down'sSyndrome in middle age, drug-induced movement disorders induced by drugswhich block CNS dopamine receptors, drug sensitivity, eczema,encephalomyelitis, endocarditis, endocrinopathy, epiglottitis,epstein-barr virus infection, erythromelalgia, extrapyramidal andcerebellar disorders, familial hematophagocytic lymphohistiocytosis,fetal thymus implant rejection, Friedreich's ataxia, functionalperipheral arterial disorders, fungal sepsis, gas gangrene, gastriculcer, glomerular nephritis, graft rejection of any organ or tissue,gram negative sepsis, gram positive sepsis, granulomas due tointracellular organisms, hairy cell leukemia, Hallerrorden-Spatzdisease, hashimoto's thyroiditis, hay fever, heart transplant rejection,hemachromatosis, hemodialysis, hemolytic uremic syndrome/thrombolyticthrombocytopenic purpura, hemorrhage, hepatitis (A), His bundlearrythmias, HIV infection/HIV neuropathy, Hodgkin's disease,hyperkinetic movement disorders, hypersensitity reactions,hypersensitivity pneumonitis, hypertension, hypokinetic movementdisorders, hypothalamic-pituitary-adrenal axis evaluation, idiopathicAddison's disease, idiopathic pulmonary fibrosis, antibody mediatedcytotoxicity, Asthenia, infantile spinal muscular atrophy, inflammationof the aorta, influenza a, ionizing radiation exposure,iridocyclitis/uveitis/optic neuritis, ischemia-reperfusion injury,ischemic stroke, juvenile rheumatoid arthritis, juvenile spinal muscularatrophy, Kaposi's sarcoma, kidney transplant rejection, legionella,leishmaniasis, leprosy, lesions of the corticospinal system, lipedema,liver transplant rejection, lymphederma, malaria, malignamt Lymphoma,malignant histiocytosis, malignant melanoma, meningitis,meningococcemia, metabolic/idiopathic diseases, migraine headache,mitochondrial multi.system disorder, mixed connective tissue disease,monoclonal gammopathy, multiple myeloma, multiple systems degenerations(Mencel Dejerine-Thomas Shi-Drager and Machado-Joseph), myastheniagravis, mycobacterium avium intracellulare, mycobacterium tuberculosis,myelodyplastic syndrome, myocardial infarction, myocardial ischemicdisorders, nasopharyngeal carcinoma, neonatal chronic lung disease,nephritis, nephrosis, neurodegenerative diseases, neurogenic I muscularatrophies, neutropenic fever, non-hodgkins lymphoma, occlusion of theabdominal aorta and its branches, occlusive arterial disorders, okt3therapy, orchitis/epidydimitis, orchitis/vasectomy reversal procedures,organomegaly, osteoporosis, pancreas transplant rejection, pancreaticcarcinoma, paraneoplastic syndrome/hypercalcemia of malignancy,parathyroid transplant rejection, pelvic inflammatory disease, perennialrhinitis, pericardial disease, peripheral atherlosclerotic disease,peripheral vascular disorders, peritonitis, pernicious anemia,pneumocystis carinii pneumonia, pneumonia, POEMS syndrome(polyneuropathy, organomegaly, endocrinopathy, monoclonal gammopathy,and skin changes syndrome), post perfusion syndrome, post pump syndrome,post-MI cardiotomy syndrome, preeclampsia, Progressive supranucleoPalsy, primary pulmonary hypertension, radiation therapy, Raynaud'sphenomenon and disease, Raynoud's disease, Refsum's disease, regularnarrow QRS tachycardia, renovascular hypertension, reperfusion injury,restrictive cardiomyopathy, sarcomas, scleroderma, senile chorea, SenileDementia of Lewy body type, seronegative arthropathies, shock, sicklecell anemia, skin allograft rejection, skin changes syndrome, smallbowel transplant rejection, solid tumors, specific arrythmias, spinalataxia, spinocerebellar degenerations, streptococcal myositis,structural lesions of the cerebellum, Subacute sclerosingpanencephalitis, Syncope, syphilis of the cardiovascular system,systemic anaphalaxis, systemic inflammatory response syndrome, systemiconset juvenile rheumatoid arthritis, T-cell or FAB ALL, Telangiectasia,thromboangitis obliterans, thrombocytopenia, toxicity, transplants,trauma/hemorrhage, type III hypersensitivity reactions, type IVhypersensitivity, unstable angina, uremia, urosepsis, urticaria,valvular heart diseases, varicose veins, vasculitis, venous diseases,venous thrombosis, ventricular fibrillation, viral and fungalinfections, vital encephalitis/aseptic meningitis, vital-associatedhemaphagocytic syndrome, Wernicke-Korsakoff syndrome, Wilson's disease,xenograft rejection of any organ or tissue, acute coronary syndromes,acute idiopathic polyneuritis, acute inflammatory demyelinatingpolyradiculoneuropathy, acute ischemia, adult Still's disease, alopeciagreata, anaphylaxis, antiphospholipid antibody syndrome, aplasticanemia, arteriosclerosis, atopic eczema, atopic dermatitis, autoimmunedermatitis, autoimmune disorder associated with streptococcus infection,autoimmune enteropathy, autoimmune hearing loss, autoimmunelymphoproliferative syndrome (ALPS), autoimmune myocarditis, autoimmunepremature ovarian failure, blepharitis, bronchiectasis, bullouspemphigoid, cardiovascular disease, catastrophic antiphospholipidsyndrome, celiac disease, cervical spondylosis, chronic ischemia,cicatricial pemphigoid, clinically isolated syndrome (CIS) with risk formultiple sclerosis, conjunctivitis, childhood onset psychiatricdisorder, chronic obstructive pulmonary disease (COPD), dacryocystitis,dermatomyositis, diabetic retinopathy, diabetes mellitus, diskherniation, disk prolaps, drug induced immune hemolytic anemia,endocarditis, endometriosis, endophthalmitis, episcleritis, erythemamultiforme, erythema multiforme major, gestational pemphigoid,Guillain-Barré syndrome (GBS), hay fever, Hughes syndrome, idiopathicParkinson's disease, idiopathic interstitial pneumonia, IgE-mediatedallergy, immune hemolytic anemia, inclusion body myositis, infectiousocular inflammatory disease, inflammatory demyelinating disease,inflammatory heart disease, inflammatory kidney disease, IPF/UIP,iritis, keratitis, keratojuntivitis sicca, Kussmaul disease orKussmaul-Meier disease, Landry's paralysis, Langerhan's cellhistiocytosis, livedo reticularis, macular degeneration, microscopicpolyangiitis, morbus bechterev, motor neuron disorders, mucous membranepemphigoid, multiple organ failure, myasthenia gravis, myelodysplasticsyndrome, myocarditis, nerve root disorders, neuropathy, non-A non-Bhepatitis, optic neuritis, osteolysis, ovarian cancer, pauciarticularJRA, peripheral artery occlusive disease (PAOD), peripheral vasculardisease (PVD), peripheral artery, disease (PAD), phlebitis,polyarteritis nodosa (or periarteritis nodosa), polychondritis,polymyalgia rheumatica, poliosis, polyarticular JRA, polyendocrinedeficiency syndrome, polymyositis, polymyalgia rheumatica (PMR),post-pump syndrome, primary Parkinsonism, prostate and rectal cancer andhematopoietic malignancies (leukemia and lymphoma), prostatitis, purered cell aplasia, primary adrenal insufficiency, recurrent neuromyelitisoptica, restenosis, rheumatic heart disease, sapho (synovitis, acne,pustulosis, hyperostosis, and osteitis), scleroderma, secondaryamyloidosis, shock lung, scleritis, sciatica, secondary adrenalinsufficiency, silicone associated connective tissue disease,sneddon-wilkinson dermatosis, spondilitis ankylosans, Stevens-Johnsonsyndrome (SJS), systemic inflammatory response syndrome, temporalarteritis, toxoplasmic retinitis, toxic epidermal necrolysis, transversemyelitis, TRAPS (tumor necrosis factor receptor, type 1 allergicreaction, type II diabetes, urticaria, usual interstitial pneumonia(UIP), vasculitis, vernal conjunctivitis, viral retinitis,Vogt-Koyanagi-Harada syndrome (VKH syndrome), wet macular degeneration,wound healing, yersinia and salmonella associated arthropathy.

In an embodiment, diseases that can be treated or diagnosed with thecompositions and methods of the invention include, but are not limitedto, primary and metastatic cancers, including carcinomas of breast,colon, rectum, lung, oropharynx, hypopharynx, esophagus, stomach,pancreas, liver, gallbladder and bile ducts, small intestine, urinarytract (including kidney, bladder and urothelium), female genital tract(including cervix, uterus, and ovaries as well as choriocarcinoma andgestational trophoblastic disease), male genital tract (includingprostate, seminal vesicles, testes and germ cell tumors), endocrineglands (including the thyroid, adrenal, and pituitary glands), and skin,as well as hemangiomas, melanomas, sarcomas (including those arisingfrom bone and soft tissues as well as Kaposi's sarcoma), tumors of thebrain, nerves, eyes, and meninges (including astrocytomas, gliomas,glioblastomas, retinoblastomas, neuromas, neuroblastomas, Schwannomas,and meningiomas), solid tumors arising from hematopoietic malignanciessuch as leukemias, and lymphomas (both Hodgkin's and non-Hodgkin'slymphomas).

In an embodiment, the antibodies of the invention or antigen-bindingportions thereof, are used to treat cancer or in the prevention orinhibition of metastases from the tumors described herein either whenused alone or in combination with radiotherapy and/or otherchemotherapeutic agents.

In another aspect the invention provides a method of treating a patientsuffering from a disorder comprising the step of administering any oneof the binding proteins disclosed herein before, concurrently, or afterthe administration of a second agent, as discussed herein. In aparticular embodiment the second agent is selected from the groupconsisting of budenoside, epidermal growth factor, corticosteroids,cyclosporin, sulfasalazine, aminosalicylates, 6-mercaptopurine,azathioprine, metronidazole, lipoxygenase inhibitors, mesalamine,olsalazine, balsalazide, antioxidants, thromboxane inhibitors, IL-1receptor antagonists, anti-IL-1β mAbs, anti-IL-6 or IL-6 receptor mAbs,growth factors, elastase inhibitors, pyridinyl-imidazole compounds,antibodies or agonists of TNF, LT, IL-1, IL-2, IL-6, IL-7, IL-8, IL-12,IL-13, IL-15, IL-16, IL-18, IL-23, EMAP-II, GM-CSF, FGF, and PDGF,antibodies of CD2, CD3, CD4, CD8, CD-19, CD25, CD28, CD30, CD40, CD45,CD69, CD90 or their ligands, methotrexate, cyclosporin, FK506,rapamycin, mycophenolate mofetil, leflunomide, NSAIDs, ibuprofen,corticosteroids, prednisolone, phosphodiesterase inhibitors, adensosineagonists, antithrombotic agents, complement inhibitors, adrenergicagents, IRAK, NIK, IKK, p38, MAP kinase inhibitors, IL-1β convertingenzyme inhibitors, TNFα converting enzyme inhibitors, T-cell signallinginhibitors, metalloproteinase inhibitors, sulfasalazine, azathioprine,6-mercaptopurines, angiotensin converting enzyme inhibitors, solublecytokine receptors, soluble p55 TNF receptor, soluble p75 TNF receptor,sIL-1RI, sIL-1RII, sIL-6R, antiinflammatory cytokines, IL-4, IL-10,IL-11, IL-13 and TGFβ.

In a particular embodiment the pharmaceutical compositions disclosedherein are administered to the patient by at least one mode selectedfrom parenteral, subcutaneous, intramuscular, intravenous,intrarticular, intrabronchial, intraabdominal, intracapsular,intracartilaginous, intracavitary, intracelial, intracerebellar,intracerebroventricular, intracolic, intracervical, intragastric,intrahepatic, intramyocardial, intraosteal, intrapelvic,intrapericardiac, intraperitoneal, intrapleural, intraprostatic,intrapulmonary, intrarectal, intrarenal, intraretinal, intraspinal,intrasynovial, intrathoracic, intrauterine, intravesical, bolus,vaginal, rectal, buccal, sublingual, intranasal, and transdermal.

One aspect of the invention provides at least one anti-idiotypicantibody to at least one binding protein of the present invention. Theanti-idiotypic antibody includes any protein or peptide containingmolecule that comprises at least a portion of an immunoglobulin moleculesuch as, but not limited to, at least one complementarily determiningregion (CDR) of a heavy or light chain or a ligand binding portionthereof, a heavy chain or light chain variable region, a heavy chain orlight chain constant region, a framework region, or any portion thereof,that can be incorporated into a binding protein of the presentinvention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A is a schematic representation of Dual Variable Domain (DVD)-Igconstructs and shows the strategy for generation of a DVD-Ig from twoparent antibodies;

FIG. 1B, is a schematic representation of constructs DVD1-Ig, DVD2-Ig,and two chimeric mono-specific antibodies from hybridoma clones 2D13.E3(anti-IL-1α) and 13F5.G5 (anti-IL-1β).

DETAILED DESCRIPTION OF THE INVENTION

This invention pertains to multivalent and/or multispecific bindingproteins that bind two or more antigens. Specifically, the inventionrelates to dual variable domain immunoglobulins (DVD-Ig), andpharmaceutical compositions thereof, as well as nucleic acids,recombinant expression vectors and host cells for making such DVD-Igs.Methods of using the DVD-Igs of the invention to detect specificantigens, either in vitro or in vivo are also encompassed by theinvention.

Unless otherwise defined herein, scientific and technical terms used inconnection with the present invention shall have the meanings that arecommonly understood by those of ordinary skill in the art. The meaningand scope of the terms should be clear, however, in the event of anylatent ambiguity, definitions provided herein take precedent over anydictionary or extrinsic definition. Further, unless otherwise requiredby context, singular terms shall include pluralities and plural termsshall include the singular. In this application, the use of “or” means“and/or” unless stated otherwise. Furthermore, the use of the term“including”, as well as other forms, such as “includes” and “included”,is not limiting. Also, terms such as “element” or “component” encompassboth elements and components comprising one unit and elements andcomponents that comprise more than one subunit unless specificallystated otherwise.

Generally, nomenclatures used in connection with, and techniques of,cell and tissue culture, molecular biology, immunology, microbiology,genetics and protein and nucleic acid chemistry and hybridizationdescribed herein are those well known and commonly used in the art. Themethods and techniques of the present invention are generally performedaccording to conventional methods well known in the art and as describedin various general and more specific references that are cited anddiscussed throughout the present specification unless otherwiseindicated. Enzymatic reactions and purification techniques are performedaccording to manufacturer's specifications, as commonly accomplished inthe art or as described herein. The nomenclatures used in connectionwith, and the laboratory procedures and techniques of, analyticalchemistry, synthetic organic chemistry, and medicinal and pharmaceuticalchemistry described herein are those well known and commonly used in theart. Standard techniques are used for chemical syntheses, chemicalanalyses, pharmaceutical preparation, formulation, and delivery, andtreatment of patients.

That the present invention may be more readily understood, select termsare defined below.

The term “polypeptide” refers to any polymeric chain of amino acids. Theterms “peptide” and “protein” are used interchangeably with the termpolypeptide and also refer to a polymeric chain of amino acids. The term“polypeptide” encompasses native or artificial proteins, proteinfragments and polypeptide analogs of a protein sequence. A polypeptidemay be monomeric or polymeric. The term “polypeptide” encompassespolypeptide and fragments and variants (including fragments of variants)thereof, unless otherwise contradicted by context. For an antigenicpolypeptide, a fragment of polypeptide optionally contains at least onecontiguous or nonlinear epitope of polypeptide. The precise boundariesof the at least one epitope fragment can be confirmed using ordinaryskill in the art. The fragment comprises at least about 5 contiguousamino acids, such as at least about 10 contiguous amino acids, at leastabout 15 contiguous amino acids, or at least about 20 contiguous aminoacids. A variant of a polypeptide is as described herein.

The term “isolated protein” or “isolated polypeptide” is a protein orpolypeptide that by virtue of its origin or source of derivation is notassociated with naturally associated components that accompany it in itsnative state; is substantially free of other proteins from the samespecies; is expressed by a cell from a different species; or does notoccur in nature. Thus, a polypeptide that is chemically synthesized orsynthesized in a cellular system different from the cell from which itnaturally originates will be “isolated” from its naturally associatedcomponents. A protein may also be rendered substantially free ofnaturally associated components by isolation, using protein purificationtechniques well known in the art.

The term “recovering” refers to the process of rendering a chemicalspecies such as a polypeptide substantially free of naturally associatedcomponents by isolation, e.g., using protein purification techniqueswell known in the art.

The term “biological activity” refers to any one or more inherentbiological properties of a molecule (whether present naturally as foundin vivo, or provided or enabled by recombinant means). Biologicalproperties include but are not limited to binding receptor; induction ofcell proliferation, inhibiting cell growth, inductions of othercytokines, induction of apoptosis, and enzymatic activity. Biologicalactivity also includes activity of an Ig molecule.

The terms “specific binding” or “specifically binding” in reference tothe interaction of an antibody, a protein, or a peptide with a secondchemical species, mean that the interaction is dependent upon thepresence of a particular structure (e.g., an antigenic determinant orepitope) on the chemical species; for example, an antibody recognizesand binds to a specific protein structure rather than to proteinsgenerally. If an antibody is specific for epitope “A”, the presence of amolecule containing epitope A (or free, unlabeled A), in a reactioncontaining labeled “A” and the antibody, will reduce the amount oflabeled A bound to the antibody.

The term “antibody” broadly refers to any immunoglobulin (Ig) moleculecomprised of four polypeptide chains, two heavy (H) chains and two light(L) chains, or any functional fragment, mutant, variant, or derivationthereof, which retains the essential epitope binding features of an Igmolecule. Such mutant, variant, or derivative antibody formats are knownin the art. Nonlimiting embodiments of which are discussed below.

In a full-length antibody, each heavy chain is comprised of a heavychain variable region (abbreviated herein as HCVR or VH) and a heavychain constant region. The heavy chain constant region is comprised ofthree domains, CH1, CH2 and CH3. Each light chain is comprised of alight chain variable region (abbreviated herein as LCVR or VL) and alight chain constant region. The light chain constant region iscomprised of one domain, CL. The VH and VL regions can be furthersubdivided into regions of hypervariability, termed complementaritydetermining regions (CDR), interspersed with regions that are moreconserved, termed framework regions (FR). Each VH and VL is composed ofthree CDRs and four FRs, arranged from amino-terminus tocarboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3,CDR3, and FR4. Immunoglobulin molecules can be of any type (e.g., IgG,IgE, IgM, IgD, IgA and IgY), class (e.g., IgG 1, IgG2, IgG 3, IgG4, IgA1and IgA2) or subclass.

The term “Fc region” is used to define the C-terminal region of animmunoglobulin heavy chain, which may be generated by papain digestionof an intact antibody. The Fc region may be a native sequence Fc regionor a variant Fc region. The Fc region of an immunoglobulin generallycomprises two constant domains, a CH2 domain and a CH3 domain, andoptionally comprises a CH4 domain. Replacements of amino acid residuesin the Fc portion to alter antibody effector function are known in theart (U.S. Pat. Nos. 5,648,260 and 5,624,821). The Fc portion of anantibody mediates several important effector functions e.g., cytokineinduction, ADCC, phagocytosis, complement dependent cytotoxicity (CDC)and half-life/clearance rate of antibody and antigen-antibody complexes.In some cases these effector functions are desirable for a therapeuticantibody but in other cases might be unnecessary or even deleterious,depending on the therapeutic objectives. Certain human IgG isotypes,particularly IgG1 and IgG3, mediate ADCC and CDC via binding to FcγRsand complement C1q, respectively. Neonatal Fc receptors (FcRn) are thecritical components determining the circulating half-life of antibodies.In still another embodiment at least one amino acid residue is replacedin the constant region of the antibody, for example the Fc region of theantibody, such that effector functions of the antibody are altered. Thedimerization of two identical heavy chains of an immunoglobulin ismediated by the dimerization of CH3 domains and is stabilized by thedisulfide bonds within the hinge region (Huber et al. (1976) Nature264:415-20; Thies et al. (1999) J. Mol. Biol. 293:67-79). Mutation ofcysteine residues within the hinge regions to prevent heavy chain-heavychain disulfide bonds will destabilize dimeration of CH3 domains.Residues responsible for CH3 dimerization have been identified(Dall'Acqua (1998) Biochem. 37:9266-73). Therefore, it is possible togenerate a monovalent half-Ig. Interestingly, these monovalent half Igmolecules have been found in nature for both IgG and IgA subclasses(Seligman (1978) Ann. Immunol. 129:855-70; Biewenga et al. (1983) Clin.Exp. Immunol. 51:395-400). The stoichiometry of FcRn: Ig Fc region hasbeen determined to be 2:1 (West et al. (2000) Biochem. 39:9698-708), andhalf Fc is sufficient for mediating FcRn binding (Kim et al. (1994) Eur.J. Immunol. 24:542-548). Mutations to disrupt the dimerization of CH3domain may not have greater adverse effect on its FcRn binding as theresidues important for CH3 dimerization are located on the innerinterface of CH3 b sheet structure, whereas the region responsible forFcRn binding is located on the outside interface of CH2-CH3 domains.However the half Ig molecule may have certain advantage in tissuepenetration due to its smaller size than that of a regular antibody. Inone embodiment at least one amino acid residue is replaced in theconstant region of the binding protein of the invention, for example theFc region, such that the dimerization of the heavy chains is disrupted,resulting in half DVD Ig molecules. The anti-inflammatory activity ofIgG is completely dependent on sialylation of the N-linked glycan of theIgG Fc fragment. The precise glycan requirements for anti-inflammatoryactivity has been determined, such that an appropriate IgG1 Fc fragmentcan be created, thereby generating a fully recombinant, sialylated IgG1Fc with greatly enhanced potency (Anthony et al. (2008) Science320:373-376).

The term “antigen-binding portion” of an antibody refers to one or morefragments of an antibody that retain the ability to specifically bind toan antigen. It has been shown that the antigen-binding function of anantibody can be performed by fragments of a full-length antibody. Suchantibody embodiments may also be bispecific, dual specific, ormulti-specific formats; specifically binding to two or more differentantigens. Examples of binding fragments encompassed within the term“antigen-binding portion” of an antibody include (i) a Fab fragment, amonovalent fragment consisting of the VL, VH, CL and CH1 domains; (ii) aF(ab′)₂ fragment, a bivalent fragment comprising two Fab fragmentslinked by a disulfide bridge at the hinge region; (iii) a Fd fragmentconsisting of the VH and CH1 domains; (iv) a Fv fragment consisting ofthe VL and VH domains of a single arm of an antibody, (v) a dAb fragment(Ward et al. (1989) Nature 341:544-546, PCT Publication WO 90/05144),which comprises a single variable domain; and (vi) an isolatedcomplementarity determining region (CDR). Furthermore, although the twodomains of the Fv fragment, VL and VH, are coded for by separate genes,they can be joined, using recombinant methods, by a synthetic linkerthat enables them to be made as a single protein chain in which the VLand VH regions pair to form monovalent molecules (known as single chainFv (scFv); see e.g., Bird et al. (1988) Science 242:423-426; and Hustonet al. (1988) Proc. Natl. Acad. Sci. USA 85:5879-5883). Such singlechain antibodies are also intended to be encompassed within the term“antigen-binding portion” of an antibody. Other forms of single chainantibodies, such as diabodies are also encompassed. Diabodies arebivalent, bispecific antibodies in which VH and VL domains are expressedon a single polypeptide chain, but using a linker that is too short toallow for pairing between the two domains on the same chain, therebyforcing the domains to pair with complementary domains of another chainand creating two antigen binding sites (see e.g., Holliger et al. (1993)Proc. Natl. Acad. Sci. USA 90:6444-6448; Poljak et al. (1994) Structure2:1121-1123). Such antibody binding portions are known in the art(Kontermann and Dubel eds., Antibody Engineering (2001) Springer-Verlag.New York. 790 pp. (ISBN 3-540-41354-5). In addition single chainantibodies also include “linear antibodies” comprising a pair of tandemFv segments (VH-CH1-VH-CH1) which, together with complementary lightchain polypeptides, form a pair of antigen binding regions (Zapata etal. (1995) Protein Eng. 8(10):1057-1062; and U.S. Pat. No. 5,641,870).

The term “multivalent binding protein” is used throughout thisspecification to denote a binding protein comprising two or more antigenbinding sites. In an embodiment, the multivalent binding protein isengineered to have the three or more antigen binding sites, and isgenerally not a naturally occurring antibody. The term “multispecificbinding protein” refers to a binding protein that binds two or morerelated or unrelated targets. Dual variable domain (DVD) bindingproteins of the invention comprise two or more antigen binding sites andare tetravalent or multivalent binding proteins. DVDs may bemonospecific, i.e., capable of binding one antigen or multispecific,i.e. capable of binding two or more antigens. DVD binding proteinscomprising two heavy chain DVD polypeptides and two light chain DVDpolypeptides are referred to as DVD-Ig. Each half of a DVD-Ig comprisesa heavy chain DVD polypeptide, and a light chain DVD polypeptide, andtwo antigen binding sites. Each binding site comprises a heavy chainvariable domain and a light chain variable domain with a total of 6 CDRsinvolved in antigen binding per antigen binding site.

The term “bispecific antibody” refers to full-length antibodies that aregenerated by quadroma technology (see Milstein and Cuello (1983) Nature305(5934):537-40), by chemical conjugation of two different monoclonalantibodies (see Staerz et al. (1985) Nature 314(6012):628-31), or byknob-into-hole or similar approaches which introduces mutations in theFc region (see Holliger et al. (1993) Proc. Natl. Acad. Sci. USA90(14):6444-8.18), resulting in multiple different immunoglobulinspecies of which only one is the functional bispecific antibody. Bymolecular function, a bispecific antibody binds one antigen (or epitope)on one of its two binding arms (one pair of HC/LC), and binds adifferent antigen (or epitope) on its second arm (a different pair ofHC/LC). By this definition, a bispecific antibody has two distinctantigen binding arms (in both specificity and CDR sequences), and ismonovalent for each antigen it binds to.

The term “dual-specific antibody” refers to full-length antibodies thatcan bind two different antigens (or epitopes) in each of its two bindingarms (a pair of HC/LC) (see PCT Publication No. WO 02/02773).Accordingly a dual-specific binding protein has two identical antigenbinding arms, with identical specificity and identical CDR sequences,and is bivalent for each antigen to which it binds.

A “functional antigen binding site” of a binding protein is one thatbinds a target antigen. The antigen binding affinity of the antigenbinding site is not necessarily as strong as the parent antibody fromwhich the antigen binding site is derived, but the ability to bindantigen must be measurable using any one of a variety of methods knownfor evaluating antibody binding to an antigen. Moreover, the antigenbinding affinity of each of the antigen binding sites of a multivalentantibody herein need not be quantitatively the same.

The term “cytokine” is a generic term for proteins released by one cellpopulation, which act on another cell population as intercellularmediators. Examples of such cytokines are lymphokines, monokines, andtraditional polypeptide hormones. Included among the cytokines aregrowth hormone such as human growth hormone, N-methionyl human growthhormone, and bovine growth hormone; parathyroid hormone; thyroxine;insulin; proinsulin; relaxin; prorelaxin; glycoprotein hormones such asfollicle stimulating hormone (FSH), thyroid stimulating hormone (TSH),and luteinizing hormone (LH); hepatic growth factor; fibroblast growthfactor; prolactin; placental lactogen; tumor necrosis factor-alpha and-beta; mullerian-inhibiting substance; mouse gonadotropin-associatedpeptide; inhibin; activin; vascular endothelial growth factor; integrin;thrombopoietin (TPO); nerve growth factors such as NGF-alpha;platelet-growth factor; placental growth factor, transforming growthfactors (TGFs) such as TGF-alpha and TGF-beta; insulin-like growthfactor-1 and -11; erythropoietin (EPO); osteoinductive factors;interferons such as interferon-alpha, -beta and -gamma colonystimulating factors (CSFs) such as macrophage-CSF (M-CSF); granulocytemacrophage-CSF (GM-CSF); and granulocyte-CSF (G-CSF); interleukins (ILs)such as IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10,IL-11, IL-12, IL-13, IL-15, IL-18, IL-21, IL-22, IL-23, and IL-33; atumor necrosis factor such as TNF-alpha or TNF-beta; and otherpolypeptide factors including LIF and kit ligand (KL). The term cytokineincludes proteins from natural sources or from recombinant cell cultureand biologically active equivalents of the native sequence cytokines.

The term “linker” is used to denote polypeptides comprising two or moreamino acid residues joined by peptide bonds and are used to link one ormore antigen binding portions. Such linker polypeptides are well knownin the art (see e.g., Holliger et al. (1993) Proc. Natl. Acad. Sci. USA90:6444-6448; Poljak et al. (1994) Structure 2:1121-1123). Exemplarylinkers include, but are not limited to, AKTTPKLEEGEFSEAR (SEQ ID NO:1); AKTTPKLEEGEFSEARV (SEQ ID NO: 2); AKTTPKLGG (SEQ ID NO: 3);SAKTTPKLGG (SEQ ID NO: 4); SAKTTP (SEQ ID NO: 5); RADAAP (SEQ ID NO: 6);RADAAPTVS (SEQ ID NO: 7); RADAAAAGGPGS (SEQ ID NO: 8); RADAAAA(G₄S)₄(SEQ ID NO: 9); SAKTTPKLEEGEFSEARV (SEQ ID NO: 10); ADAAP (SEQ ID NO:11); ADAAPTVSIFPP (SEQ ID NO: 12); TVAAP (SEQ ID NO: 13); TVAAPSVFIFPP(SEQ ID NO: 14); QPKAAP (SEQ ID NO: 15); QPKAAPSVTLFPP (SEQ ID NO: 16);AKTTPP (SEQ ID NO: 17); AKTTPPSVTPLAP (SEQ ID NO: 18); AKTTAP (SEQ IDNO: 19); AKTTAPSVYPLAP (SEQ ID NO: 20); ASTKGP (SEQ ID NO: 21);ASTKGPSVFPLAP (SEQ ID NO: 22), GGGGSGGGGSGGGGS (SEQ ID NO: 23);GENKVEYAPALMALS (SEQ ID NO: 24); GPAKELTPLKEAKVS (SEQ ID NO: 25);GHEAAAVMQVQYPAS (SEQ ID NO: 26), TVAAPSVFIFPPTVAAPSVFIFPP (SEQ ID NO:27); and ASTKGPSVFPLAPASTKGPSVFPLAP (SEQ ID NO: 28).

An immunoglobulin constant domain refers to a heavy or light chainconstant domain. Human IgG heavy chain and light chain constant domainamino acid sequences are known in the art.

The term “monoclonal antibody” or “mAb” refers to an antibody obtainedfrom a population of substantially homogeneous antibodies, i.e., theindividual antibodies comprising the population are identical except forpossible naturally occurring mutations that may be present in minoramounts. Monoclonal antibodies are highly specific, being directedagainst a single antigen. Furthermore, in contrast to polyclonalantibody preparations that typically include different antibodiesdirected against different determinants (epitopes), each mAb is directedagainst a single determinant on the antigen. The modifier “monoclonal”is not to be construed as requiring production of the antibody by anyparticular method.

The term “human antibody” includes antibodies having variable andconstant regions derived from human germline immunoglobulin sequences.The human antibodies of the invention may include amino acid residuesnot encoded by human germline immunoglobulin sequences (e.g., mutationsintroduced by random or site-specific mutagenesis in vitro or by somaticmutation in vivo), for example in the CDRs and in particular CDR3.However, the term “human antibody” is not intended to include antibodiesin which CDR sequences derived from the germline of another mammalianspecies, such as a mouse, have been grafted onto human frameworksequences.

The term “recombinant human antibody” includes all human antibodies thatare prepared, expressed, created or isolated by recombinant means, suchas antibodies expressed using a recombinant expression vectortransfected into a host cell (described further in Section II C, below),antibodies isolated from a recombinant, combinatorial human antibodylibrary (Hoogenboom (1997) TIB Tech. 15:62-70; Azzazy and Highsmith(2002) Clin. Biochem. 35:425-445; Gavilondo and Larrick (2002)BioTechniques 29:128-145; Hoogenboom and Chames (2000) Immunol. Today21:371-378), antibodies isolated from an animal (e.g., a mouse) that istransgenic for human immunoglobulin genes (see, Taylor et al. (1992)Nucl. Acids Res. 20:6287-6295; Kellermann and Green (2002) Current Opin.Biotechnol. 13:593-597; Little et al. (2000) Immunol. Today 21:364-370)or antibodies prepared, expressed, created or isolated by any othermeans that involves splicing of human immunoglobulin gene sequences toother DNA sequences. Such recombinant human antibodies have variable andconstant regions derived from human germline immunoglobulin sequences.In certain embodiments, however, such recombinant human antibodies aresubjected to in vitro mutagenesis (or, when an animal transgenic forhuman Ig sequences is used, in vivo somatic mutagenesis) and thus theamino acid sequences of the VH and VL regions of the recombinantantibodies are sequences that, while derived from and related to humangermline VH and VL sequences, may not naturally exist within the humanantibody germline repertoire in vivo.

An “affinity matured” antibody is an antibody with one or morealterations in one or more CDRs thereof which result an improvement inthe affinity of the antibody for antigen, compared to a parent antibodywhich does not possess those alteration(s). Exemplary affinity maturedantibodies will have nanomolar or even picomolar affinities for thetarget antigen. Affinity matured antibodies are produced by proceduresknown in the art. Marks et al. (1992) Bio/Technology 10:779-783describes affinity maturation by VH and VL domain shuffling. Randommutagenesis of CDR and/or framework residues is described by: Barbas etal. (1994) Proc Nat. Acad. Sci. USA 91:3809-3813; Schier et al. (1995)Gene 169:147-155; Yelton et al. (1995) J. Immunol. 155:1994-2004;Jackson et al. (1995) J. Immunol. 154(7):3310-9; Hawkins et al. (1992)J. Mol. Biol. 226:889-896 and selective mutation at selectivemutagenesis positions, contact or hypermutation positions with anactivity enhancing amino acid residue as described in U.S. Pat. No.6,914,128.

The term “chimeric antibody” refers to antibodies which comprise heavyand light chain variable region sequences from one species and constantregion sequences from another species, such as antibodies having murineheavy and light chain variable regions linked to human constant regions.

The term “CDR-grafted antibody” refers to antibodies which compriseheavy and light chain variable region sequences from one species but inwhich the sequences of one or more of the CDR regions of VH and/or VLare replaced with CDR sequences of another species, such as antibodieshaving murine heavy and light chain variable regions in which one ormore of the murine CDRs (e.g., CDR3) has been replaced with human CDRsequences.

The term “humanized antibody” refers to antibodies which comprise heavyand light chain variable region sequences from a non-human species(e.g., a mouse) but in which at least a portion of the VH and/or VLsequence has been altered to be more “human-like”, i.e., more similar tohuman germline variable sequences. One type of humanized antibody is aCDR-grafted antibody, in which non-human CDR sequences are introducedinto human VH and VL sequences to replace the corresponding human CDRsequences. Also “humanized antibody” is an antibody or a variant,derivative, analog or fragment thereof which immunospecifically binds toan antigen of interest and which comprises a framework (FR) regionhaving substantially the amino acid sequence of a human antibody and acomplementary determining region (CDR) having substantially the aminoacid sequence of a non-human antibody. The term “substantially” in thecontext of a CDR refers to a CDR having an amino acid sequence at least80%, at least 85%, at least 90%, at least 95%, at least 98% or at least99% identical to the amino acid sequence of a non-human antibody CDR. Ahumanized antibody comprises substantially all of at least one, andtypically two, variable domains (Fab, Fab′, F(ab′)2, FabC, Fv) in whichall or substantially all of the CDR regions correspond to those of anon-human immunoglobulin (i.e., donor antibody) and all or substantiallyall of the framework regions are those of a human immunoglobulinconsensus sequence. In an embodiment, a humanized antibody alsocomprises at least a portion of an immunoglobulin constant region (Fc),typically that of a human immunoglobulin. In some embodiments, ahumanized antibody contains both the light chain as well as at least thevariable domain of a heavy chain. The antibody also may include the CH1,hinge, CH2, CH3, and CH4 regions of the heavy chain. In someembodiments, a humanized antibody only contains a humanized light chain.In some embodiments, a humanized antibody only contains a humanizedheavy chain. In specific embodiments, a humanized antibody only containsa humanized variable domain of a light chain and/or humanized heavychain.

The terms “Kabat numbering”, “Kabat definitions” and “Kabat labeling”are used interchangeably herein. These terms, which are recognized inthe art, refer to a system of numbering amino acid residues which aremore variable (i.e., hypervariable) than other amino acid residues inthe heavy and light chain variable regions of an antibody, or an antigenbinding portion thereof (Kabat et al. (1971) Ann. NY Acad. Sci.190:382-391 and Kabat et al. (1991) Sequences of Proteins ofImmunological Interest, Fifth Edition, U.S. Department of Health andHuman Services, NIH Publication No. 91-3242). For the heavy chainvariable region, the hypervariable region ranges from amino acidpositions 31 to 35 for CDR1, amino acid positions 50 to 65 for CDR2, andamino acid positions 95 to 102 for CDR3. For the light chain variableregion, the hypervariable region ranges from amino acid positions 24 to34 for CDR1, amino acid positions 50 to 56 for CDR2, and amino acidpositions 89 to 97 for CDR3.

The term “CDR” refers to the complementarity determining region withinantibody variable sequences. There are three CDRs in each of thevariable regions of the heavy chain and the light chain, which aredesignated CDR1, CDR2 and CDR3, for each of the variable regions. Theterm “CDR set” refers to a group of three CDRs that occur in a singlevariable region that binds the antigen. The exact boundaries of theseCDRs have been defined differently according to different systems. Thesystem described by Kabat (Kabat et al., Sequences of Proteins ofImmunological Interest (National Institutes of Health, Bethesda, Md.(1987) and (1991)) not only provides an unambiguous residue numberingsystem applicable to any variable region of an antibody, but alsoprovides precise residue boundaries defining the three CDRs. These CDRsmay be referred to as Kabat CDRs. Chothia and coworkers (Chothia andLesk (1987) J. Mol. Biol. 196:901-917 and Chothia et al. (1989) Nature342:877-883) found that certain sub-portions within Kabat CDRs adoptnearly identical peptide backbone conformations, despite having greatdiversity at the level of amino acid sequence. These sub-portions weredesignated as L1, L2 and L3 or H1, H2 and H3 where the “L” and the “H”designates the light chain and the heavy chains regions, respectively.These regions may be referred to as Chothia CDRs, which have boundariesthat overlap with Kabat CDRs. Other boundaries defining CDRs overlappingwith the Kabat CDRs have been described by Padlan (1995) FASEB J.9:133-139 and MacCallum (1996) J. Mol. Biol. 262(5):732-45). Still otherCDR boundary definitions may not strictly follow one of the hereinsystems, but will nonetheless overlap with the Kabat CDRs, although theymay be shortened or lengthened in light of prediction or experimentalfindings that particular residues or groups of residues or even entireCDRs do not significantly impact antigen binding. The methods usedherein may utilize CDRs defined according to any of these systems,although certain embodiments use Kabat or Chothia defined CDRs.

The term “framework” or “framework sequence” refers to the remainingsequences of a variable region minus the CDRs. Because the exactdefinition of a CDR sequence can be determined by different systems, themeaning of a framework sequence is subject to correspondingly differentinterpretations. The six CDRs (CDR-L1, -L2, and -L3 of light chain andCDR-H1, -H2, and -H3 of heavy chain) also divide the framework regionson the light chain and the heavy chain into four sub-regions (FR1, FR2,FR3 and FR4) on each chain, in which CDR1 is positioned between FR1 andFR2, CDR2 between FR2 and FR3, and CDR3 between FR3 and FR4. Withoutspecifying the particular sub-regions as FR1, FR2, FR3 or FR4, aframework region, as referred by others, represents the combined FR'swithin the variable region of a single, naturally occurringimmunoglobulin chain. An FR represents one of the four sub-regions, andFRs represents two or more of the four sub-regions constituting aframework region.

The term “germline antibody gene” or “gene fragment” refers to animmunoglobulin sequence encoded by non-lymphoid cells that have notundergone the maturation process that leads to genetic rearrangement andmutation for expression of a particular immunoglobulin. (See, e.g.,Shapiro et al. (2002) Crit. Rev. Immunol. 22(3):183-200; Marchalonis etal. (2001) Adv. Exp. Med. Biol. 484:13-30). One of the advantagesprovided by various embodiments of the present invention stems from therecognition that germline antibody genes are more likely than matureantibody genes to conserve essential amino acid sequence structurescharacteristic of individuals in the species, hence less likely to berecognized as from a foreign source when used therapeutically in thatspecies.

The term “neutralizing” refers to counteracting the biological activityof an antigen when a binding protein specifically binds the antigen. Inan embodiment, the neutralizing binding protein binds the cytokine andreduces its biologically activity by at least about 20%, 40%, 60%, 80%,85% or more.

The term “activity” includes activities such as the binding specificityand affinity of a DVD-Ig for two or more antigens.

The term “epitope” includes any polypeptide determinant capable ofspecific binding to an immunoglobulin or T-cell receptor. In certainembodiments, epitope determinants include chemically active surfacegroupings of molecules such as amino acids, sugar side chains,phosphoryl, or sulfonyl, and, in certain embodiments, may have specificthree dimensional structural characteristics, and/or specific chargecharacteristics. An epitope is a region of an antigen that is bound byan antibody. An epitope thus consists of the amino acid residues of aregion of an antigen (or fragment thereof) known to bind to thecomplementary site on the specific binding partner. An antigenicfragment can contain more than one epitope. In certain embodiments, anantibody is said to specifically bind an antigen when it recognizes itstarget antigen in a complex mixture of proteins and/or macromolecules.Antibodies are said to “bind to the same epitope” if the antibodiescross-compete (one prevents the binding or modulating effect of theother). In addition structural definitions of epitopes (overlapping,similar, identical) are informative, but functional definitions areoften more relevant as they encompass structural (binding) andfunctional (modulation, competition) parameters.

The term “surface plasmon resonance” refers to an optical phenomenonthat allows for the analysis of real-time biospecific interactions bydetection of alterations in protein concentrations within a biosensormatrix, for example using the BIAcore® system (BIAcore International AB,a GE Healthcare company, Uppsala, Sweden and Piscataway, N.J.). Forfurther descriptions, see Jönsson et al. (1993) Ann. Biol. Clin.51:19-26; Jönsson et al. (1991) Biotechniques 11:620-627; Johnsson etal. (1995) J. Mol. Recognit. 8:125-131; and Johnnson, et al. (1991)Anal. Biochem. 198:268-277.

The term “K_(on)” refers to the on rate constant for association of abinding protein (e.g., an antibody) to the antigen to form the, e.g.,antibody/antigen complex as is known in the art. The “Kon” also is knownby the terms “association rate constant”, or “ka”, as usedinterchangeably herein. This value indicating the binding rate of anantibody to its target antigen or the rate of complex formation betweenan antibody and antigen also is shown by the equation below:Antibody (“Ab”)+Antigen (“Ag”)→Ab−Ag.

The term “K_(off)” refers to the off rate constant for dissociation, or“dissociation rate constant”, of a binding protein (e.g., an antibody)from the, e.g., antibody/antigen complex as is known in the art. The“Koff” also is known by the terms “dissociation rate constant” or “kd”as used interchangeably herein. This value indicates the dissociationrate of an antibody from its target antigen or separation of Ab−Agcomplex over time into free antibody and antigen as shown by theequation below:Ab+Ag←Ab−Ag.

The term “K_(D)” refers to the “equilibrium dissociation constant”, or“KD,” as used interchangeably herein, refer to the value obtained in atitration measurement at equilibrium, or by dividing the dissociationrate constant (koff) by the association rate constant (kon). Theassociation rate constant, the dissociation rate constant and theequilibrium dissociation constant are used to represent the bindingaffinity of an antibody to an antigen. Methods for determiningassociation and dissociation rate constants are well known in the art.Using fluorescence-based techniques offers high sensitivity and theability to examine samples in physiological buffers at equilibrium.Other experimental approaches and instruments such as a BIAcore®(biomolecular interaction analysis) assay can be used (e.g., instrumentavailable from BIAcore International AB, a GE Healthcare company,Uppsala, Sweden). Additionally, a KinExA® (Kinetic Exclusion Assay)assay, available from Sapidyne Instruments (Boise, Id.) can also beused.

“Label” and “detectable label” mean a moiety attached to a specificbinding partner, such as an antibody or an analyte, e.g., to render thereaction between members of a specific binding pair, such as an antibodyand an analyte, detectable, and the specific binding partner, e.g.,antibody or analyte, so labeled is referred to as “detectably labeled.”Thus, the term “labeled binding protein” refers to a protein with alabel incorporated that provides for the identification of the bindingprotein. In an embodiment, the label is a detectable marker that canproduce a signal that is detectable by visual or instrumental means,e.g., incorporation of a radiolabeled amino acid or attachment to apolypeptide of biotinyl moieties that can be detected by marked avidin(e.g., streptavidin containing a fluorescent marker or enzymaticactivity that can be detected by optical or colorimetric methods).Examples of labels for polypeptides include, but are not limited to, thefollowing: radioisotopes or radionuclides (e.g., ³H, ¹⁴C, ³⁵S, ⁹⁰Y,⁹⁹Tc, ¹¹¹In, ¹²⁵I, ¹³¹I, ¹⁷⁷Lu, ¹⁶⁶Ho, or ¹⁵³Sm); chromogens,fluorescent labels (e.g., FITC, rhodamine, lanthanide phosphors),enzymatic labels (e.g., horseradish peroxidase, luciferase, alkalinephosphatase); chemiluminescent markers; biotinyl groups; predeterminedpolypeptide epitopes recognized by a secondary reporter (e.g., leucinezipper pair sequences, binding sites for secondary antibodies, metalbinding domains, and epitope tags); and magnetic agents, such asgadolinium chelates. Representative examples of labels commonly employedfor immunoassays include moieties that produce light, e.g., acridiniumcompounds, and moieties that produce fluorescence, e.g., fluorescein.Other labels are described herein. In this regard, the moiety itself maynot be detectably labeled but may become detectable upon reaction withyet another moiety. Use of “detectably labeled” is intended to encompassthe latter type of detectable labeling.

The term “conjugate” refers to a binding protein, such as an antibody,chemically linked to a second chemical moiety, such as a therapeutic orcytotoxic agent. The term “agent” denotes a chemical compound, a mixtureof chemical compounds, a biological macromolecule, or an extract madefrom biological materials. In an embodiment, the therapeutic orcytotoxic agents include, but are not limited to, pertussis toxin,taxol, cytochalasin B, gramicidin D, ethidium bromide, emetine,mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicin,doxorubicin, daunorubicin, dihydroxy anthracin dione, mitoxantrone,mithramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids,procaine, tetracaine, lidocaine, propranolol, and puromycin and analogsor homologs thereof. When employed in the context of an immunoassay, theconjugate antibody may be a detectably labeled antibody used as thedetection antibody.

The terms “crystal” and “crystallized” refer to a binding protein (e.g.,an antibody), or antigen binding portion thereof, that exists in theform of a crystal. Crystals are one form of the solid state of matter,which is distinct from other forms such as the amorphous solid state orthe liquid crystalline state. Crystals are composed of regular,repeating, three-dimensional arrays of atoms, ions, molecules (e.g.,proteins such as antibodies), or molecular assemblies (e.g.,antigen/antibody complexes). These three-dimensional arrays are arrangedaccording to specific mathematical relationships that arewell-understood in the field. The fundamental unit, or building block,that is repeated in a crystal is called the asymmetric unit. Repetitionof the asymmetric unit in an arrangement that conforms to a given,well-defined crystallographic symmetry provides the “unit cell” of thecrystal. Repetition of the unit cell by regular translations in allthree dimensions provides the crystal. See Giege and Ducruix (1999)Crystallization of Nucleic Acids and Proteins, a Practical Approach, 2ndea., pp. 20 1-16, Oxford University Press, New York, N.Y.

The term “polynucleotide” means a polymeric form of two or morenucleotides, either ribonucleotides or deoxynucleotides or a modifiedform of either type of nucleotide. The term includes single and doublestranded forms of DNA.

The term “isolated polynucleotide” shall mean a polynucleotide (e.g., ofgenomic, cDNA, or synthetic origin, or some combination thereof) that,by virtue of its origin, the “isolated polynucleotide” is not associatedwith all or a portion of a polynucleotide with which the “isolatedpolynucleotide” is found in nature; is operably linked to apolynucleotide that it is not linked to in nature; or does not occur innature as part of a larger sequence.

The term “vector”, is intended to refer to a nucleic acid moleculecapable of transporting another nucleic acid to which it has beenlinked. One type of vector is a “plasmid”, which refers to a circulardouble stranded DNA loop into which additional DNA segments may beligated. Another type of vector is a viral vector, wherein additionalDNA segments may be ligated into the viral genome. Certain vectors arecapable of autonomous replication in a host cell into which they areintroduced (e.g., bacterial vectors having a bacterial origin ofreplication and episomal mammalian vectors). Other vectors (e.g.,non-episomal mammalian vectors) can be integrated into the genome of ahost cell upon introduction into the host cell, and thereby arereplicated along with the host genome. Moreover, certain vectors arecapable of directing the expression of genes to which they areoperatively linked. Such vectors are referred to herein as “recombinantexpression vectors” (or simply, “expression vectors”). In general,expression vectors of utility in recombinant DNA techniques are often inthe form of plasmids. In the present specification, “plasmid” and“vector” may be used interchangeably as the plasmid is the most commonlyused form of vector. However, the invention is intended to include suchother forms of expression vectors, such as viral vectors (e.g.,replication defective retroviruses, adenoviruses and adeno-associatedviruses), which serve equivalent functions.

The term “operably linked” refers to a juxtaposition wherein thecomponents described are in a relationship permitting them to functionin their intended manner. A control sequence “operably linked” to acoding sequence is ligated in such a way that expression of the codingsequence is achieved under conditions compatible with the controlsequences. “Operably linked” sequences include both expression controlsequences that are contiguous with the gene of interest and expressioncontrol sequences that act in trans or at a distance to control the geneof interest. The term “expression control sequence” refers topolynucleotide sequences which are necessary to effect the expressionand processing of coding sequences to which they are ligated. Expressioncontrol sequences include appropriate transcription initiation,termination, promoter and enhancer sequences; efficient RNA processingsignals such as splicing and polyadenylation signals; sequences thatstabilize cytoplasmic mRNA; sequences that enhance translationefficiency (i.e., Kozak consensus sequence); sequences that enhanceprotein stability; and when desired, sequences that enhance proteinsecretion. The nature of such control sequences differs depending uponthe host organism; in prokaryotes, such control sequences generallyinclude promoter, ribosomal binding site, and transcription terminationsequence; in eukaryotes, generally, such control sequences includepromoters and transcription termination sequence. The term “controlsequences” is intended to include components whose presence is essentialfor expression and processing, and can also include additionalcomponents whose presence is advantageous, for example, leader sequencesand fusion partner sequences.

“Transformation”, refers to any process by which exogenous DNA enters ahost cell. Transformation may occur under natural or artificialconditions using various methods well known in the art. Transformationmay rely on any known method for the insertion of foreign nucleic acidsequences into a prokaryotic or eukaryotic host cell. The method isselected based on the host cell being transformed and may include, butis not limited to, viral infection, electroporation, lipofection, andparticle bombardment. Such “transformed” cells include stablytransformed cells in which the inserted DNA is capable of replicationeither as an autonomously replicating plasmid or as part of the hostchromosome. They also include cells which transiently express theinserted DNA or RNA for limited periods of time.

The term “recombinant host cell” (or simply “host cell”), is intended torefer to a cell into which exogenous DNA has been introduced. In anembodiment, the host cell comprises two or more (e.g., multiple) nucleicacids encoding antibodies, such as the host cells described in U.S. Pat.No. 7,262,028, for example. Such terms are intended to refer not only tothe particular subject cell, but also to the progeny of such a cell.Because certain modifications may occur in succeeding generations due toeither mutation or environmental influences, such progeny may not, infact, be identical to the parent cell, but are still included within thescope of the term “host cell”. In an embodiment, host cells includeprokaryotic and eukaryotic cells selected from any of the Kingdoms oflife. In another embodiment, eukaryotic cells include protist, fungal,plant and animal cells. In another embodiment, host cells include butare not limited to the prokaryotic cell line E. Coli; mammalian celllines CHO, HEK 293, COS, NS0, SP2 and PER.C6; the insect cell line Sf9;and the fungal cell Saccharomyces cerevisiae.

Standard techniques may be used for recombinant DNA, oligonucleotidesynthesis, and tissue culture and transformation (e.g., electroporation,lipofection). Enzymatic reactions and purification techniques may beperformed according to manufacturer's specifications or as commonlyaccomplished in the art or as described herein. The foregoing techniquesand procedures may be generally performed according to conventionalmethods well known in the art and as described in various general andmore specific references that are cited and discussed throughout thepresent specification. See e.g., Sambrook et al. (1989) MolecularCloning: A Laboratory Manual (2d ed., Cold Spring Harbor LaboratoryPress, Cold Spring Harbor, N.Y.).

“Transgenic organism”, as known in the art, refers to an organism havingcells that contain a transgene, wherein the transgene introduced intothe organism (or an ancestor of the organism) expresses a polypeptidenot naturally expressed in the organism. A “transgene” is a DNAconstruct, which is stably and operably integrated into the genome of acell from which a transgenic organism develops, directing the expressionof an encoded gene product in one or more cell types or tissues of thetransgenic organism.

The terms “regulate” and “modulate” refer to a change or an alterationin the activity of a molecule of interest (e.g., the biological activityof a cytokine). Modulation may be an increase or a decrease in themagnitude of a certain activity or function of the molecule of interest.Exemplary activities and functions of a molecule include, but are notlimited to, binding characteristics, enzymatic activity, cell receptoractivation, and signal transduction.

Correspondingly, the term “modulator” is a compound capable of changingor altering an activity or function of a molecule of interest (e.g., thebiological activity of a cytokine). For example, a modulator may causean increase or decrease in the magnitude of a certain activity orfunction of a molecule compared to the magnitude of the activity orfunction observed in the absence of the modulator. In certainembodiments, a modulator is an inhibitor, which decreases the magnitudeof at least one activity or function of a molecule. Exemplary inhibitorsinclude, but are not limited to, proteins, peptides, antibodies,peptibodies, carbohydrates or small organic molecules. Peptibodies aredescribed, e.g., in PCT Publication No. WO01/83525.

The term “agonist”, refers to a modulator that, when contacted with amolecule of interest, causes an increase in the magnitude of a certainactivity or function of the molecule compared to the magnitude of theactivity or function observed in the absence of the agonist. Particularagonists of interest may include, but are not limited to, polypeptides,nucleic acids, carbohydrates, or any other molecules that bind to theantigen.

The term “antagonist” or “inhibitor” refers to a modulator that, whencontacted with a molecule of interest causes a decrease in the magnitudeof a certain activity or function of the molecule compared to themagnitude of the activity or function observed in the absence of theantagonist. Particular antagonists of interest include those that blockor modulate the biological or immunological activity of the antigen.Antagonists and inhibitors of antigens may include, but are not limitedto, proteins, nucleic acids, carbohydrates, or any other molecules,which bind to the antigen.

The term “effective amount” refers to the amount of a therapy which issufficient to reduce or ameliorate the severity and/or duration of adisorder or one or more symptoms thereof, inhibit or prevent theadvancement of a disorder, cause regression of a disorder, inhibit orprevent the recurrence, development, onset or progression of one or moresymptoms associated with a disorder, detect a disorder, or enhance orimprove the prophylactic or therapeutic effect(s) of another therapy(e.g., prophylactic or therapeutic agent).

The terms “patient” and “subject” may be used interchangeably herein torefer to an animal, such as a mammal, including a primate (for example,a human, a monkey, and a chimpanzee), a non-primate (for example, a cow,a pig, a camel, a llama, a horse, a goat, a rabbit, a sheep, a hamster,a guinea pig, a cat, a dog, a rat, a mouse, and a whale), a bird (e.g.,a duck or a goose), and a shark. Preferably, the patient or subject is ahuman, such as a human being treated or assessed for a disease, disorderor condition, a human at risk for a disease, disorder or condition, ahuman having a disease, disorder or condition, and/or human beingtreated for a disease, disorder or condition.

The term “sample” is used in its broadest sense. A “biological sample”includes, but is not limited to, any quantity of a substance from aliving thing or formerly living thing. Such living things include, butare not limited to, humans, mice, rats, monkeys, dogs, rabbits and otheranimals. Such substances include, but are not limited to, blood (e.g.,whole blood), plasma, serum, urine, amniotic fluid, synovial fluid,endothelial cells, leukocytes, monocytes, other cells, organs, tissues,bone marrow, lymph nodes and spleen.

“Component,” “components,” and “at least one component,” refer generallyto a capture antibody, a detection or conjugate antibody, a control, acalibrator, a series of calibrators, a sensitivity panel, a container, abuffer, a diluent, a salt, an enzyme, a co-factor for an enzyme, adetection reagent, a pretreatment reagent/solution, a substrate (e.g.,as a solution), a stop solution, and the like that can be included in akit for assay of a test sample, such as a patient urine, serum or plasmasample, in accordance with the methods described herein and othermethods known in the art. Thus, in the context of the presentdisclosure, “at least one component,” “component,” and “components” caninclude a polypeptide or other analyte as above, such as a compositioncomprising an analyte such as polypeptide, which is optionallyimmobilized on a solid support, such as by binding to an anti-analyte(e.g., anti-polypeptide) antibody. Some components can be in solution orlyophilized for reconstitution for use in an assay.

“Control” refers to a composition known to not contain analyte(“negative control”) or to contain analyte (“positive control”). Apositive control can comprise a known concentration of analyte.“Control,” “positive control,” and “calibrator” may be usedinterchangeably herein to refer to a composition comprising a knownconcentration of analyte. A “positive control” can be used to establishassay performance characteristics and is a useful indicator of theintegrity of reagents (e.g., analytes).

“Predetermined cutoff” and “predetermined level” refer generally to anassay cutoff value that is used to assessdiagnostic/prognostic/therapeutic efficacy results by comparing theassay results against the predetermined cutoff/level, where thepredetermined cutoff/level already has been linked or associated withvarious clinical parameters (e.g., severity of disease,progression/nonprogression/improvement, etc.). While the presentdisclosure may provide exemplary predetermined levels, it is well-knownthat cutoff values may vary depending on the nature of the immunoassay(e.g., antibodies employed, etc.). It further is well within theordinary skill of one in the art to adapt the disclosure herein forother immunoassays to obtain immunoassay-specific cutoff values forthose other immunoassays based on this disclosure. Whereas the precisevalue of the predetermined cutoff/level may vary between assays,correlations as described herein (if any) should be generallyapplicable.

“Pretreatment reagent,” e.g., lysis, precipitation and/or solubilizationreagent, as used in a diagnostic assay as described herein is one thatlyses any cells and/or solubilizes any analyte that is/are present in atest sample. Pretreatment is not necessary for all samples, as describedfurther herein. Among other things, solubilizing the analyte (e.g.,polypeptide of interest) may entail release of the analyte from anyendogenous binding proteins present in the sample. A pretreatmentreagent may be homogeneous (not requiring a separation step) orheterogeneous (requiring a separation step). With use of a heterogeneouspretreatment reagent there is removal of any precipitated analytebinding proteins from the test sample prior to proceeding to the nextstep of the assay.

“Quality control reagents” in the context of immunoassays and kitsdescribed herein, include, but are not limited to, calibrators,controls, and sensitivity panels. A “calibrator” or “standard” typicallyis used (e.g., one or more, such as a plurality) in order to establishcalibration (standard) curves for interpolation of the concentration ofan analyte, such as an antibody or an analyte. Alternatively, a singlecalibrator, which is near a predetermined positive/negative cutoff, canbe used. Multiple calibrators (i.e., more than one calibrator or avarying amount of calibrator(s)) can be used in conjunction so as tocomprise a “sensitivity panel.”

“Risk” refers to the possibility or probability of a particular eventoccurring either presently or at some point in the future. “Riskstratification” refers to an array of known clinical risk factors thatallows physicians to classify patients into a low, moderate, high orhighest risk of developing a particular disease, disorder or condition.

“Specific” and “specificity” in the context of an interaction betweenmembers of a specific binding pair (e.g., an antigen (or fragmentthereof) and an antibody (or antigenically reactive fragment thereof))refer to the selective reactivity of the interaction. The phrase“specifically binds to” and analogous phrases refer to the ability ofantibodies (or antigenically reactive fragments thereof) to bindspecifically to analyte (or a fragment thereof) and not bindspecifically to other entities.

“Specific binding partner” is a member of a specific binding pair. Aspecific binding pair comprises two different molecules, whichspecifically bind to each other through chemical or physical means.Therefore, in addition to antigen and antibody specific binding pairs ofcommon immunoassays, other specific binding pairs can include biotin andavidin (or streptavidin), carbohydrates and lectins, complementarynucleotide sequences, effector and receptor molecules, cofactors andenzymes, enzyme inhibitors and enzymes, and the like. Furthermore,specific binding pairs can include members that are analogs of theoriginal specific binding members, for example, an analyte-analog.Immunoreactive specific binding members include antigens, antigenfragments, and antibodies, including monoclonal and polyclonalantibodies as well as complexes, fragments, and variants (includingfragments of variants) thereof, whether isolated or recombinantlyproduced.

“Variant” means a polypeptide that differs from a given polypeptide(e.g., IL-18, BNP, NGAL or HIV polypeptide or anti-polypeptide antibody)in amino acid sequence by the addition (e.g., insertion), deletion, orconservative substitution of amino acids, but that retains thebiological activity of the given polypeptide (e.g., a variant IL-18 cancompete with anti-IL-18 antibody for binding to IL-18). A conservativesubstitution of an amino acid, i.e., replacing an amino acid with adifferent amino acid of similar properties (e.g., hydrophilicity anddegree and distribution of charged regions) is recognized in the art astypically involving a minor change. These minor changes can beidentified, in part, by considering the hydropathic index of aminoacids, as understood in the art (see, e.g., Kyte et al. (1982) J. Mol.Biol. 157:105-132). The hydropathic index of an amino acid is based on aconsideration of its hydrophobicity and charge. It is known in the artthat amino acids of similar hydropathic indexes can be substituted andstill retain protein function. In one aspect, amino acids havinghydropathic indexes of ±2 are substituted. The hydrophilicity of aminoacids also can be used to reveal substitutions that would result inproteins retaining biological function. A consideration of thehydrophilicity of amino acids in the context of a peptide permitscalculation of the greatest local average hydrophilicity of thatpeptide, a useful measure that has been reported to correlate well withantigenicity and immunogenicity (see, e.g., U.S. Pat. No. 4,554,101).Substitution of amino acids having similar hydrophilicity values canresult in peptides retaining biological activity, for exampleimmunogenicity, as is understood in the art. In one aspect,substitutions are performed with amino acids having hydrophilicityvalues within ±2 of each other. Both the hydrophobicity index and thehydrophilicity value of amino acids are influenced by the particularside chain of that amino acid. Consistent with that observation, aminoacid substitutions that are compatible with biological function areunderstood to depend on the relative similarity of the amino acids, andparticularly the side chains of those amino acids, as revealed by thehydrophobicity, hydrophilicity, charge, size, and other properties.“Variant” also can be used to describe a polypeptide or fragment thereofthat has been differentially processed, such as by proteolysis,phosphorylation, or other post-translational modification, yet retainsits biological activity or antigen reactivity, e.g., the ability to bindto IL-18. The term “variant” encompasses fragments of a variant unlessotherwise contradicted by context.

I. Generation of DVD Binding Protein

The invention pertains to Dual Variable Domain binding proteins thatbind one or more targets and methods of making the same. In anembodiment, the binding protein comprises a polypeptide chain, whereinsaid polypeptide chain comprises VD1-(X1)n-VD2-C-(X2)n, wherein VD1 is afirst variable domain, VD2 is a second variable domain, C is a constantdomain, X1 represents an amino acid or polypeptide, X2 represents an Fcregion and n is 0 or 1. The binding protein of the invention can begenerated using various techniques. The invention provides expressionvectors, host cell and methods of generating the binding protein.

A. Generation of Parent Monoclonal Antibodies

The variable domains of the DVD binding protein can be obtained fromparent antibodies, including polyclonal and mAbs that bind antigens ofinterest. These antibodies may be naturally occurring or may begenerated by recombinant technology.

MAbs can be prepared using a wide variety of techniques known in the artincluding the use of hybridoma, recombinant, and phage displaytechnologies, or a combination thereof. For example, mAbs can beproduced using hybridoma techniques including those known in the art andtaught, for example, in Harlow et al. (1988) Antibodies: A LaboratoryManual, (Cold Spring Harbor Laboratory Press, 2nd ed.); Hammerling etal. (1981) in: Monoclonal Antibodies and T-Cell Hybridomas 563-681(Elsevier, N.Y.). The term “monoclonal antibody” is not limited toantibodies produced through hybridoma technology. The term “monoclonalantibody” refers to an antibody that is derived from a single clone,including any eukaryotic, prokaryotic, or phage clone, and not themethod by which it is produced. Hybridomas are selected, cloned andfurther screened for desirable characteristics, including robusthybridoma growth, high antibody production and desirable antibodycharacteristics, as discussed in Example 1 below. Hybridomas may becultured and expanded in vivo in syngeneic animals, in animals that lackan immune system, e.g., nude mice, or in cell culture in vitro. Methodsof selecting, cloning and expanding hybridomas are well known to thoseof ordinary skill in the art. In a particular embodiment, the hybridomasare mouse hybridomas. In another embodiment, the hybridomas are producedin a non-human, non-mouse species such as rats, sheep, pigs, goats,cattle or horses. In another embodiment, the hybridomas are humanhybridomas, in which a human non-secretory myeloma is fused with a humancell expressing an antibody that bind a specific antigen.

Recombinant mAbs are also generated from single, isolated lymphocytesusing a procedure referred to in the art as the selected lymphocyteantibody method (SLAM), as described in U.S. Pat. No. 5,627,052; PCTPublication No. WO 92/02551; and Babcock et al. (1996) Proc. Natl. Acad.Sci. USA 93:7843-7848. In this method, single cells secreting antibodiesof interest, e.g., lymphocytes derived from an immunized animal, areidentified, and, heavy- and light-chain variable region cDNAs arerescued from the cells by reverse transcriptase-PCR and these variableregions can then be expressed, in the context of appropriateimmunoglobulin constant regions (e.g., human constant regions), inmammalian host cells, such as COS or CHO cells. The host cellstransfected with the amplified immunoglobulin sequences, derived from invivo selected lymphocytes, can then undergo further analysis andselection in vitro, for example by panning the transfected cells toisolate cells expressing antibodies to the antigen of interest. Theamplified immunoglobulin sequences further can be manipulated in vitro,such as by in vitro affinity maturation methods such as those describedin PCT Publication Nos. WO 97/29131 and WO 00/56772.

Monoclonal antibodies are also produced by immunizing a non-human animalcomprising some, or all, of the human immunoglobulin locus with anantigen of interest. In an embodiment, the non-human animal is aXENOMOUSE transgenic mouse, an engineered mouse strain that compriseslarge fragments of the human immunoglobulin loci and is deficient inmouse antibody production. See, e.g., Green et al. (1994) Nature Genet.7:13-21 and U.S. Pat. Nos. 5,916,771; 5,939,598; 5,985,615; 5,998,209;6,075,181; 6,091,001; 6,114,598 and 6,130,364. See also PCT PublicationNos. WO 91/10741; WO 94/02602; WO 96/34096; WO 96/33735; WO 98/16654; WO98/24893; WO 98/50433; WO 99/45031; WO 99/53049; WO 00 09560; and WO00/037504. The XENOMOUSE transgenic mouse produces an adult-like humanrepertoire of fully human antibodies, and generates antigen-specifichuman monoclonal antibodies. The XENOMOUSE transgenic mouse containsapproximately 80% of the human antibody repertoire through introductionof megabase sized, germline configuration YAC fragments of the humanheavy chain loci and κ (kappa) light chain loci. See Mendez et al.(1997) Nature Genet. 15:146-156; Green and Jakobovits (1998) J. Exp.Med. 188:483-495.

In vitro methods also can be used to make the parent antibodies, whereinan antibody library is screened to identify an antibody having thedesired binding specificity. Methods for such screening of recombinantantibody libraries are well known in the art and include methodsdescribed in, for example, U.S. Pat. No. 5,223,409; PCT Publication Nos.WO 92/18619; WO 91/17271; WO 92/20791; WO 92/15679; WO 93/01288; WO92/01047; WO 92/09690; and WO 97/29131; Fuchs et al. (1991)Bio/Technology 9:1370-1372; Hay et al. (1992) Hum. Antibod. Hybridomas3:81-85; Huse et al. (1989) Science 246:1275-1281; McCafferty et al.(1990) Nature 348:552-554; Griffiths et al. (1993) EMBO J. 12:725-734;Hawkins et al. (1992) J. Mol. Biol. 226:889-896; Clackson et al. (1991)Nature 352:624-628; Gram et al. (1992) Proc. Natl. Acad. Sci. USA89:3576-3580; Garrad et al. (1991) Bio/Technology 9:1373-1377;Hoogenboom et al. (1991) Nucl. Acid Res. 19:4133-4137; and Barbas et al.(1991) Proc. Natl. Acad. Sci. USA 88:7978-7982; and US Publication No.20030186374.

Parent antibodies of the present invention can also be generated usingvarious phage display methods known in the art. In phage displaymethods, functional antibody domains are displayed on the surface ofphage particles that carry the polynucleotide sequences encoding them.In a particular, such phage can be utilized to display antigen-bindingdomains expressed from a repertoire or combinatorial antibody library(e.g., human or murine). Phage expressing an antigen binding domain thatbinds the antigen of interest can be selected or identified withantigen, e.g., using labeled antigen or antigen bound or captured to asolid surface or bead. Phage used in these methods are typicallyfilamentous phage including fd and M13 binding domains expressed fromphage with Fab, Fv or disulfide stabilized Fv antibody domainsrecombinantly fused to either the phage gene III or gene VIII protein.Examples of phage display methods that can be used to make theantibodies of the present invention include those disclosed in Brinkmanet al. (1995) J. Immunol. Methods 182:41-50; Ames et al. (1995) J.Immunol. Methods 184:177-186; Kettleborough et al. (1994) Eur. J.Immunol. 24:952-958; Persic et al. (1997) Gene 187 9-18; Burton et al.(1994) Advances Immunol. 57:191-280; PCT Publication Nos. WO 90/02809;WO 91/10737; WO 92/01047; WO 92/18619; WO 93/11236; WO 95/15982; and WO95/20401; and U.S. Pat. Nos. 5,698,426; 5,223,409; 5,403,484; 5,580,717;5,427,908; 5,750,753; 5,821,047; 5,571,698; 5,427,908; 5,516,637;5,780,225; 5,658,727; 5,733,743; and 5,969,108.

After phage selection, the antibody coding regions from the phage can beisolated and used to generate whole antibodies including humanantibodies or any other desired antigen binding fragment, and expressedin any desired host, including mammalian cells, insect cells, plantcells, yeast, and bacteria, e.g., as described in detail below. Forexample, techniques to recombinantly produce Fab, Fab′ and F(ab′)2fragments can also be employed using methods known in the art such asthose disclosed in PCT Publication No. WO 92/22324; Mullinax et al.,(1992) BioTechniques 12(6):864-869; and Sawai et al. (1995) AJRI34:26-34; and Better et al. (1988) Science 240:1041-1043. Examples oftechniques which can be used to produce single-chain Fvs and antibodiesinclude those described in U.S. Pat. Nos. 4,946,778 and 5,258,498;Huston et al. (1991) Methods Enzymol. 203:46-88; Shu et al. (1993) Proc.Natl. Acad. Sci. USA 90:7995-7999; and Skerra et al. (1988) Science240:1038-1040.

Alternative to screening of recombinant antibody libraries by phagedisplay, other methodologies known in the art for screening largecombinatorial libraries can be applied to the identification of parentantibodies. One type of alternative expression system is one in whichthe recombinant antibody library is expressed as RNA-protein fusions, asdescribed in PCT Publication No. WO 98/31700 by Szostak and Roberts, andin Roberts and Szostak (1997) Proc. Natl. Acad. Sci. USA 94:12297-12302.In this system, a covalent fusion is created between an mRNA and thepeptide or protein that it encodes by in vitro translation of syntheticmRNAs that carry puromycin, a peptidyl acceptor antibiotic, at their 3′end. Thus, a specific mRNA can be enriched from a complex mixture ofmRNAs (e.g., a combinatorial library) based on the properties of theencoded peptide or protein, e.g., antibody, or portion thereof, such asbinding of the antibody, or portion thereof, to the dual specificityantigen. Nucleic acid sequences encoding antibodies, or portionsthereof, recovered from screening of such libraries can be expressed byrecombinant means as described herein (e.g., in mammalian host cells)and, moreover, can be subjected to further affinity maturation by eitheradditional rounds of screening of mRNA-peptide fusions in whichmutations have been introduced into the originally selected sequence(s),or by other methods for affinity maturation in vitro of recombinantantibodies, as described herein.

In another approach the parent antibodies can also be generated usingyeast display methods known in the art. In yeast display methods,genetic methods are used to tether antibody domains to the yeast cellwall and display them on the surface of yeast. In particular, such yeastcan be utilized to display antigen-binding domains expressed from arepertoire or combinatorial antibody library (e.g., human or murine).Examples of yeast display methods that can be used to make the parentantibodies include those disclosed in U.S. Pat. No. 6,699,658.

The antibodies described herein can be further modified to generate CDRgrafted and humanized parent antibodies. CDR-grafted parent antibodiescomprise heavy and light chain variable region sequences from a humanantibody wherein one or more of the CDR regions of V_(H) and/or V_(L)are replaced with CDR sequences of murine antibodies that bind antigenof interest. A framework sequence from any human antibody may serve asthe template for CDR grafting. However, straight chain replacement ontosuch a framework often leads to some loss of binding affinity to theantigen. The more homologous a human antibody is to the original murineantibody, the less likely the possibility that combining the murine CDRswith the human framework will introduce distortions in the CDRs thatcould reduce affinity. Therefore, in an embodiment, the human variableframework that is chosen to replace the murine variable framework apartfrom the CDRs have at least a 65% sequence identity with the murineantibody variable region framework. In an embodiment, the human andmurine variable regions apart from the CDRs have at least 70% sequenceidentify. In a particular embodiment, that the human and murine variableregions apart from the CDRs have at least 75% sequence identity. Inanother embodiment, the human and murine variable regions apart from theCDRs have at least 80% sequence identity. Methods for producing suchantibodies are known in the art (see EP Patent No. EP 239,400; PCTPublication No. WO 91/09967; U.S. Pat. Nos. 5,225,539; 5,530,101; and5,585,089), veneering or resurfacing (EP Patent Nos. EP 592,106 and EP519,596; Padlan (1991) Mol. Immunol. 28(4/5):489-498; Studnicka et al.(1994) Protein Engin. 7(6):805-814; Roguska et al. (1994) Proc. Natl.Acad. Sci. USA 91:969-973), and chain shuffling (U.S. Pat. No.5,565,352); and anti-idiotypic antibodies.

Humanized antibodies are antibody molecules from non-human speciesantibody that binds the desired antigen having one or morecomplementarity determining regions (CDRs) from the non-human speciesand framework regions from a human immunoglobulin molecule. Known humanIg sequences are disclosed, e.g.,www.ncbi.nlm.nih.gov/entrez-/query.fcgi; www.atcc.org/phage/hdb.html;www.sciquest.com/; www.abcam.com/;www.antibodyresource.com/onlinecomp.html;www.public.iastate.eduabout.pedro/research_tools.html;www.mgen.uni-heidelberg.de/SD/IT/IT.html;www.whfreeman.com/immunology/CH-05/kuby05.htm;www.library.thinkquest.org/12429/Immune/Antibody.html;www.hhmi.org/grants/lectures/1996/vlab/;www.path.cam.ac.uk/.about.mrc7/m-ikeimages.html;www.antibodyresource.com/;mcb.harvard.edu/BioLinks/Immunology.html.www.immunologylink.com/;pathbox.wustl.edu/.about.hcenter/index.-html;www.biotech.ufl.edu/.about.hcl/; www.pebio.com/pa/340913/340913.html-;www.nal.usda.gov/awic/pubs/antibody/;www.m.ehime-u.acjp/.about.yasuhito-/Elisa.html;www.biodesign.com/table.asp; www.icnet.uk/axp/facs/davies/lin-ks.html;www.biotech.ufl.edu/.about.fccl/protocol.html;www.isac-net.org/sites_geo.html;aximtl.imt.uni-marburg.de/.about.rek/AEP-Start.html;baserv.uci.kun.nl/.about.jraats/links1.html;www.recab.uni-hd.de/immuno.bme.nwu.edu/;www.mrc-cpe.cam.ac.uk/imt-doc/public/INTRO.html;www.ibt.unam.mx/vir/V_mice.html; imgt.cnusc.fr:8104/;www.biochem.ucl.ac.uk/.about.martin/abs/index.html;antibody.bath.ac.uk/; abgen.cvm.tamu.edu/lab/wwwabgen.html;www.unizh.ch/.about.honegger/AHOseminar/Slide01.html;www.cryst.bbk.ac.uk/.about.ubcg07s/;www.nimr.mrc.ac.uk/CC/ccaewg/ccaewg.htm;www.path.cam.ac.uk/.about.mrc7/humanisation/TAHHP.html;www.ibt.unam.mx/vir/structure/stat_aim.html;www.biosci.missouri.edu/smithgp/index.html;www.cryst.bioc.cam.ac.uk/.abo-ut.fmolina/Web-pages/Pept/spottech.html;www.jerini.de/frroducts.htm; www.patents.ibm.com/ibm.html.Kabat et al.,Sequences of Proteins of Immunological Interest, U.S. Dept. Health(1983). Such imported sequences can be used to reduce immunogenicity orreduce, enhance or modify binding, affinity, on-rate, off-rate, avidity,specificity, half-life, or any other suitable characteristic, as knownin the art.

Framework residues in the human framework regions may be substitutedwith the corresponding residue from the CDR donor antibody to alter,e.g., improve, antigen binding. These framework substitutions areidentified by methods well known in the art, e.g., by modeling of theinteractions of the CDR and framework residues to identify frameworkresidues important for antigen binding and sequence comparison toidentify unusual framework residues at particular positions. (See, e.g.,U.S. Pat. No. 5,585,089; Riechmann et al. (1988) Nature 332:323.Three-dimensional immunoglobulin models are commonly available and arefamiliar to those skilled in the art. Computer programs are availablewhich illustrate and display probable three-dimensional conformationalstructures of selected candidate immunoglobulin sequences. Inspection ofthese displays permits analysis of the likely role of the residues inthe functioning of the candidate immunoglobulin sequence, i.e., theanalysis of residues that influence the ability of the candidateimmunoglobulin to bind its antigen. In this way, FR residues can beselected and combined from the consensus and import sequences so thatthe desired antibody characteristic, such as increased affinity for thetarget antigen(s), is achieved. In general, the CDR residues aredirectly and most substantially involved in influencing antigen binding.Antibodies can be humanized using a variety of techniques known in theart, such as but not limited to those described in Jones et al. (1986)Nature 321:522; Verhoeyen et al. (1988) Science 239:1534; Sims et al.(1993) J. Immunol. 151:2296; Chothia and Lesk (1987) J. Mol. Biol.196:901; Carter et al. (1992) Proc. Natl. Acad. Sci. USA. 89:4285;Presta et al. (1993) J. Immunol. 151:2623; Padlan (1991) Mol. Immunol.28(4/5):489-498; Studnicka et al. (1994) Prot. Engin. 7(6):805-814;Roguska et al. (1994) Proc. Natl. Acad. Sci. USA 91:969-973; PCTPublication No. WO 91/09967, Int. Applic. Nos. PCT/US98/16280;US96/18978; US91/09630; US91/05939; US94/01234; GB89/01334; GB91/01134;GB92/01755; PCT Publication Nos. WO90/14443; WO90/14424; WO90/14430; EUPatent Nos. EP 229,246; EP 592,106; EP 519,596; EP 239,400; U.S. Pat.Nos. 5,565,332; 5,723,323; 5,976,862; 5,824,514; 5,817,483; 5,814,476;5,763,192; 5,723,323; 5,766,886; 5,714,352; 6,204,023; 6,180,370;5,693,762; 5,530,101; 5,585,089; 5,225,539; and 4,816,567.

B. Criteria for Selecting Parent Monoclonal Antibodies

An embodiment of the invention pertains to selecting parent antibodieswith at least one or more properties desired in the DVD-Ig molecule. Inan embodiment, the desired property is selected from one or moreantibody parameters. In another embodiment, the antibody parameters areselected from the group consisting of antigen specificity, affinity toantigen, potency, biological function, epitope recognition, stability,solubility, production efficiency, immunogenicity, pharmacokinetics,bioavailability, tissue cross reactivity, and orthologous antigenbinding.

B1. Affinity to Antigen

The desired affinity of a therapeutic mAb may depend upon the nature ofthe antigen, and the desired therapeutic end-point. In an embodiment,monoclonal antibodies have higher affinities (Kd=0.01−0.50 pM) whenblocking a cytokine-cytokine receptor interaction as such interactionare usually high affinity interactions (e.g., <pM−<nM ranges). In suchinstances, the mAb affinity for its target should be equal to or betterthan the affinity of the cytokine (ligand) for its receptor. On theother hand, mAb with lesser affinity (>nM range) could betherapeutically effective e.g., in clearing circulating potentiallypathogenic proteins e.g., monoclonal antibodies that bind to, sequester,and clear circulating species of A-β amyloid. In other instances,reducing the affinity of an existing high affinity mAb by site-directedmutagenesis or using a mAb with lower affinity for its target could beused to avoid potential side-effects e.g., a high affinity mAb maysequester/neutralize all of its intended target, thereby completelydepleting/eliminating the function(s) of the targeted protein. In thisscenario, a low affinity mAb may sequester/neutralize a fraction of thetarget that may be responsible for the disease symptoms (thepathological or over-produced levels), thus allowing a fraction of thetarget to continue to perform its normal physiological function(s).Therefore, it may be possible to reduce the Kd to adjust dose and/orreduce side-effects. The affinity of the parental mAb might play a rolein appropriately targeting cell surface molecules to achieve desiredtherapeutic out-come. For example, if a target is expressed on cancercells with high density and on normal cells with low density, a loweraffinity mAb will bind a greater number of targets on tumor cells thannormal cells, resulting in tumor cell elimination via ADCC or CDC, andtherefore might have therapeutically desirable effects. Thus selecting amAb with desired affinity may be relevant for both soluble and surfacetargets.

Signaling through a receptor upon interaction with its ligand may dependupon the affinity of the receptor-ligand interaction. Similarly, it isconceivable that the affinity of a mAb for a surface receptor coulddetermine the nature of intracellular signaling and whether the mAb maydeliver an agonist or an antagonist signal. The affinity-based nature ofmAb-mediated signaling may have an impact of its side-effect profile.Therefore, the desired affinity and desired functions of therapeuticmonoclonal antibodies need to be determined carefully by in vitro and invivo experimentation.

The desired Kd of a binding protein (e.g., an antibody) may bedetermined experimentally depending on the desired therapeutic outcome.In an embodiment, parent antibodies with affinity (Kd) for a particularantigen equal to, or better than, the desired affinity of the DVD-Ig forthe same antigen are selected. The parent antibodies for a given DVD-Igmolecule can be the same antibody or different antibodies. The antigenbinding affinity and kinetics are assessed by Biacore or another similartechnique. In one embodiment, each parent antibody has a dissociationconstant (Kd) to its antigen selected from the group consisting of: atmost about 10⁻⁷ M; at most about 10⁻⁸ M; at most about 10⁻⁹ M; at mostabout 10⁻¹⁰ M; at most about 10⁻¹¹ M; at most about 10⁻¹² M; and at most10⁻¹³M. First parent antibody from which VD1 is obtained and secondparent antibody from which VD2 is obtained may have similar or differentaffinity (K_(D)) for the respective antigen. Each parent antibody has anon rate constant (Kon) to the antigen selected from the group consistingof: at least about 10²M⁻¹s⁻¹; at least about 10³M⁻¹s⁻¹; at least about10⁴M⁻¹s⁻¹; at least about 10⁵M⁻¹s⁻¹; and at least about 10⁶M⁻¹s⁻¹, asmeasured by surface plasmon resonance. The first parent antibody fromwhich VD1 is obtained and the second parent antibody from which VD2 isobtained may have similar or different on rate constant (Kon) for therespective antigen. In one embodiment, each parent antibody has an offrate constant (Koff) to the antigen selected from the group consistingof: at most about 10⁻³s⁻¹; at most about 10⁻⁴s⁻¹; at most about 10⁻⁵s⁻¹;and at most about 10⁻⁶s⁻¹, as measured by surface plasmon resonance. Thefirst parent antibody from which VD1 is obtained and the second parentantibody from which VD2 is obtained may have similar or different offrate constants (Koff) for the respective antigen.

B2. Potency

The desired affinity/potency of parental monoclonal antibodies willdepend on the desired therapeutic outcome. For example, forreceptor-ligand (R-L) interactions the affinity (kd) is equal to orbetter than the R-L kd (pM range). For simple clearance of a pathologiccirculating protein, the kd could be in low nM range e.g., clearance ofvarious species of circulating A-β peptide. In addition, the kd willalso depend on whether the target expresses multiple copies of the sameepitope e.g., a mAb targeting conformational epitope in Aβ oligomers.

Where VD1 and VD2 bind the same antigen, but distint epitopes, theDVD-Ig will contain 4 binding sites for the same antigen, thusincreasing avidity and thereby the apparent kd of the DVD-Ig. In anembodiment, parent antibodies with equal or lower kd than that desiredin the DVD-Ig are chosen. The affinity considerations of a parental mAbmay also depend upon whether the DVD-Ig contains four or more identicalantigen binding sites (i.e; a DVD-Ig from a single mAb). In this case,the apparent kd would be greater than the mAb due to avidity. SuchDVD-Igs can be employed for cross-linking surface receptor, increaseneutralization potency, enhance clearance of pathological proteins etc.

In an embodiment parent antibodies with neutralization potency forspecific antigen equal to or better than the desired neutralizationpotential of the DVD-Ig for the same antigen are selected. Theneutralization potency can be assessed by a target-dependent bioassaywhere cells of appropriate type produce a measurable signal (i.e.,proliferation or cytokine production) in response to target stimulation,and target neutralization by the mAb can reduce the signal in adose-dependent manner.

B3. Biological Functions

Monoclonal antibodies can perform potentially several functions. Some ofthese functions are listed in Table 1. These functions can be assessedby both in vitro assays (e.g., cell-based and biochemical assays) and invivo animal models.

TABLE 1 Some Potential Applications For Therapeutic Antibodies Target(Class) Mechanism of Action (target) Soluble Neutralization of activity(e.g., a cytokine) (cytokines, other) Enhance clearance (e.g., Aβoligomers) Increase half-life (e.g., GLP 1) Cell Surface Agonist (e.g.,GLP1 R; EPO R; etc.) (Receptors, other) Antagonist (e.g., integrins;etc.) Cytotoxic (CD 20; etc.) Protein deposits Enhanceclearance/degradation (e.g., Aβ plaques, amyloid deposits)

MAbs with distinct functions described in the examples herein in Table 1can be selected to achieve desired therapeutic outcomes. Two or moreselected parent monoclonal antibodies can then be used in DVD-Ig formatto achieve two distinct functions in a single DVD-Ig molecule. Forexample, a DVD-Ig can be generated by selecting a parent mAb thatneutralizes function of a specific cytokine, and selecting a parent mAbthat enhances clearance of a pathological protein. Similarly, two parentmonoclonal antibodies that recognize two different cell surfacereceptors can be selected, one mAb with an agonist function on onereceptor and the other mAb with an antagonist function on a differentreceptor. These two selected monoclonal antibodies each with a distinctfunction can be used to construct a single DVD-Ig molecule that willpossess the two distinct functions (agonist and antagonist) of theselected monoclonal antibodies in a single molecule. Similarly, twoantagonistic monoclonal antibodies to cell surface receptors eachblocking binding of respective receptor ligands (e.g., EGF and IGF) canbe used in a DVD-Ig format. Conversely, an antagonistic anti-receptormAb (e.g., anti-EGFR) and a neutralizing anti-soluble mediator (e.g.,anti-IGF1/2) mAb can be selected to make a DVD-Ig.

B4. Epitope Recognition

Different regions of proteins may perform different functions. Forexample specific regions of a cytokine interact with the cytokinereceptor to bring about receptor activation whereas other regions of theprotein may be required for stabilizing the cytokine. In this instanceone may select a mAb that binds specifically to the receptor interactingregion(s) on the cytokine and thereby block cytokine-receptorinteraction. In some cases, for example certain chemokine receptors thatbind multiple ligands, a mAb that binds to the epitope (region onchemokine receptor) that interacts with only one ligand can be selected.In other instances, monoclonal antibodies can bind to epitopes on atarget that are not directly responsible for physiological functions ofthe protein, but binding of a mAb to these regions could eitherinterfere with physiological functions (steric hindrance) or alter theconformation of the protein such that the protein cannot function (mAbto receptors with multiple ligand which alter the receptor conformationsuch that none of the ligand can bind). Anti-cytokine monoclonalantibodies that do not block binding of the cytokine to its receptor,but block signal transduction have also been identified (e.g., 125-2H,an anti-IL-18 mAb).

Examples of epitopes and mAb functions include, but are not limited to,blocking Receptor-Ligand (R-L) interaction (neutralizing mAb that bindsR-interacting site); steric hindrance resulting in diminished or noR-binding. An Ab can bind the target at a site other than a receptorbinding site, but still interferes with receptor binding and functionsof the target by inducing conformational change and eliminating function(e.g., Xolair), binding to R but blocking signaling (125-2H).

In an embodiment, the parental mAb needs to target the appropriateepitope for maximum efficacy. Such epitope should be conserved in theDVD-Ig. The binding epitope of a mAb can be determined by severalapproaches, including co-crystallography, limited proteolysis ofmAb-antigen complex plus mass spectrometric peptide mapping (Legros etal. (2000) Protein Sci. 9:1002-10), phage displayed peptide libraries(O'Connor et al. (2005) J. Immunol. Methods 299:21-35), as well asmutagenesis (Wu et al. (2003) J. Immunol. 170:5571-7).

B5. Physicochemical and Pharmaceutical Properties

Therapeutic treatment with antibodies often requires administration ofhigh doses, often several mg/kg (due to a low potency on a mass basis asa consequence of a typically large molecular weight). In order toaccommodate patient compliance and to adequately address chronic diseasetherapies and outpatient treatment, subcutaneous (s.c.) or intramuscular(i.m.) administration of therapeutic mAbs is desirable. For example, themaximum desirable volume for s.c. administration is ˜1.0 mL, andtherefore, concentrations of >100 mg/mL are desirable to limit thenumber of injections per dose. In an embodiment, the therapeuticantibody is administered in one dose. The development of suchformulations is constrained, however, by protein-protein interactions(e.g., aggregation, which potentially increases immunogenicity risks)and by limitations during processing and delivery (e.g., viscosity).Consequently, the large quantities required for clinical efficacy andthe associated development constraints limit full exploitation of thepotential of antibody formulation and s.c. administration in high-doseregimens. It is apparent that the physicochemical and pharmaceuticalproperties of a protein molecule and the protein solution are of utmostimportance, e.g., stability, solubility and viscosity features.

B5.1. Stability:

A “stable” antibody formulation is one in which the antibody thereinessentially retains its physical stability and/or chemical stabilityand/or biological activity upon storage. Stability can be measured at aselected temperature for a selected time period. In an embodiment, theantibody in the formulation is stable at room temperature (about 30° C.)or at 40° C. for at least 1 month and/or stable at about 2-8° C. for atleast 1 year for at least 2 years. Furthermore, in an embodiment, theformulation is stable following freezing (to, e.g., −70° C.) and thawingof the formulation, hereinafter referred to as a “freeze/thaw cycle.” Inanother example, a “stable” formulation may be one wherein less thanabout 10% and less than about 5% of the protein is present as anaggregate in the formulation.

A DVD-Ig that is stable in vitro at various temperatures for an extendedtime period is desirable. One can achieve this by rapid screening ofparental mAbs that are stable in vitro at elevated temperature, e.g., at40° C. for 2-4 weeks, and then assess stability. During storage at 2-8°C., the protein reveals stability for at least 12 months, e.g., at least24 months. Stability (% of monomeric, intact molecule) can be assessedusing various techniques such as cation exchange chromatography, sizeexclusion chromatography, SDS-PAGE, as well as bioactivity testing. Fora more comprehensive list of analytical techniques that may be employedto analyze covalent and conformational modifications see Jones (1993)Analytical methods for the assessment of protein formulations anddelivery systems. In: Cleland, J. L.; Langer, R., editors. Formulationand delivery of peptides and proteins, 1^(st) edition, Washington, ACS,pg. 22-45; and Pearlman and Nguyen (1990) Analysis of protein drugs. In:Lee, V. H., editor. Peptide and protein drug delivery, 1st edition, NewYork, Marcel Dekker, Inc., pg. 247-301.

Heterogeneity and aggregate formation: stability of the antibody may besuch that the formulation may reveal less than about 10%, and, in anembodiment, less than about 5%, in another embodiment, less than about2%, or, in an embodiment, within the range of 0.5% to 1.5% or less inthe GMP antibody material that is present as aggregate. Size exclusionchromatography is a method that is sensitive, reproducible, and veryrobust in the detection of protein aggregates.

In addition to low aggregate levels, the antibody must, in anembodiment, be chemically stable. Chemical stability may be determinedby ion exchange chromatography (e.g., cation or anion exchangechromatography), hydrophobic interaction chromatography, or othermethods such as isoelectric focusing or capillary electrophoresis. Forinstance, chemical stability of the antibody may be such that afterstorage of at least 12 months at 2-8° C. the peak representingunmodified antibody in a cation exchange chromatography may increase notmore than 20%, in an embodiment, not more than 10%, or, in anotherembodiment, not more than 5% as compared to the antibody solution priorto storage testing.

In an embodiment, the parent antibodies display structural integrity;correct disulfide bond formation, and correct folding: Chemicalinstability due to changes in secondary or tertiary structure of anantibody may impact antibody activity. For instance, stability asindicated by activity of the antibody may be such that after storage ofat least 12 months at 2-8° C. the activity of the antibody may decreasenot more than 50%, in an embodiment not more than 30%, or even not morethan 10%, or in an embodiment not more than 5% or 1% as compared to theantibody solution prior to storage testing. Suitable antigen-bindingassays can be employed to determine antibody activity.

B5.2. Solubility

The “solubility” of a mAb correlates with the production of correctlyfolded, monomeric IgG. The solubility of the IgG may therefore beassessed by HPLC. For example, soluble (monomeric) IgG will give rise toa single peak on the HPLC chromatograph, whereas insoluble (e.g.,multimeric and aggregated) will give rise to a plurality of peaks. Aperson skilled in the art will therefore be able to detect an increaseor decrease in solubility of an IgG using routine HPLC techniques. For amore comprehensive list of analytical techniques that may be employed toanalyze solubility (see Jones (1993) Dep. Chem. Biochem. Eng., Univ.Coll. London, London, UK. Editor(s): Shamlou, P. Ayazi. Process.Solid-Liq. Suspensions, 93-117. Publisher: Butterworth-Heinemann,Oxford, UK and Pearlman and Nguyen (1990) Advances Parenteral Sci.4:247-301). Solubility of a therapeutic mAb is critical for formulatingto high concentration often required for adequate dosing. As outlinedherein, solubilities of >100 mg/mL may be required to accommodateefficient antibody dosing. For instance, antibody solubility may be notless than about 5 mg/mL in early research phase, in an embodiment notless than about 25 mg/mL in advanced process science stages, or in anembodiment not less than about 100 mg/mL, or in an embodiment not lessthan about 150 mg/mL. It is obvious to a person skilled in the art thatthe intrinsic properties of a protein molecule are important thephysico-chemical properties of the protein solution, e.g., stability,solubility, viscosity. However, a person skilled in the art willappreciate that a broad variety of excipients exist that may be used asadditives to beneficially impact the characteristics of the finalprotein formulation. These excipients may include: (i) liquid solvents,cosolvents (e.g., alcohols such as ethanol); (ii) buffering agents(e.g., phosphate, acetate, citrate, amino acid buffers); (iii) sugars orsugar alcohols (e.g., sucrose, trehalose, fructose, raffinose, mannitol,sorbitol, dextrans); (iv) surfactants (e.g., polysorbate 20, 40, 60, 80,poloxamers); (v) isotonicity modifiers (e.g., salts such as NaCl,sugars, sugar alcohols); and (vi) others (e.g., preservatives, chelatingagents, antioxidants, chelating substances (e.g., EDTA), biodegradablepolymers, carrier molecules (e.g., HSA, PEGs)

Viscosity is a parameter of high importance with regard to antibodymanufacture and antibody processing (e.g.,diafiltration/ultrafiltration), fill-finish processes (pumping aspects,filtration aspects) and delivery aspects (syringeability, sophisticateddevice delivery). Low viscosities enable the liquid solution of theantibody having a higher concentration. This enables the same dose maybe administered in smaller volumes. Small injection volumes inhere theadvantage of lower pain on injection sensations, and the solutions notnecessarily have to be isotonic to reduce pain on injection in thepatient. The viscosity of the antibody solution may be such that atshear rates of 100 (1/s) antibody solution viscosity is below 200 mPa s,in an embodiment below 125 mPa s, in another embodiment below 70 mPa s,and in yet another embodiment below 25 mPa s or even below 10 mPa s.

B 5.3. Production Efficiency

The generation of a DVD-Ig that is efficiently expressed in mammaliancells, such as Chinese hamster ovary cells (CHO), will in an embodimentrequire two parental monoclonal antibodies which are themselvesexpressed efficiently in mammalian cells. The production yield from astable mammalian line (i.e., CHO) should be above about 0.5 g/L, in anembodiment above about 1 g/L, and in another embodiment in the range ofabout 2 to about 5 g/L or more (Kipriyanov and Little (1999) Mol.Biotechnol. 12:173-201; Carroll and Al-Rubeai (2004) Expert Opin. BiolTher. 4:1821-9).

Production of antibodies and Ig fusion proteins in mammalian cells isinfluenced by several factors. Engineering of the expression vector viaincorporation of strong promoters, enhancers and selection markers canmaximize transcription of the gene of interest from an integrated vectorcopy. The identification of vector integration sites that are permissivefor high levels of gene transcription can augment protein expressionfrom a vector (Wurm et al. (2004) Nature Biotech. 22(11):1393-1398).Furthermore, levels of production are affected by the ratio of antibodyheavy and light chains and various steps in the process of proteinassembly and secretion (Jiang et al. (2006) Biotechnol. Progr.22(1):313-8).

B 6. Immunogenicity

Administration of a therapeutic mAb may result in certain incidence ofan immune response (i.e., the formation of endogenous antibodiesdirected against the therapeutic mAb). Potential elements that mightinduce immunogenicity should be analyzed during selection of theparental monoclonal antibodies, and steps to reduce such risk can betaken to optimize the parental monoclonal antibodies prior to DVD-Igconstruction. Mouse-derived antibodies have been found to be highlyimmunogenic in patients. The generation of chimeric antibodies comprisedof mouse variable and human constant regions presents a logical nextstep to reduce the immunogenicity of therapeutic antibodies (Morrisonand Schlom (1990) Import. Adv. Oncol. 3-18). Alternatively,immunogenicity can be reduced by transferring murine CDR sequences intoa human antibody framework (reshaping/CDR grafting/humanization), asdescribed for a therapeutic antibody by Riechmann et al. (1988) Nature332:32327. Another method is referred to as “resurfacing” or“veneering”, starting with the rodent variable light and heavy domains,only surface-accessible framework amino acids are altered to human ones,while the CDR and buried amino acids remain from the parental rodentantibody (Roguska et al. (1996) Protein Engineer. 9:895-904). In anothertype of humanization, instead of grafting the entire CDRs, one techniquegrafts only the “specificity-determining regions” (SDRs), defined as thesubset of CDR residues that are involved in binding of the antibody toits target (Kashmiri et al. (2005) Methods 36(1):25-34. Thisnecessitates identification of the SDRs either through analysis ofavailable three-dimensional structures of antibody-target complexes ormutational analysis of the antibody CDR residues to determine whichinteract with the target. Alternatively, fully human antibodies may havereduced immunogenicity compared to murine, chimeric or humanizedantibodies.

Another approach to reduce the immunogenicity of therapeutic antibodiesis the elimination of certain specific sequences that are predicted tobe immunogenic. In one approach, after a first generation biologic hasbeen tested in humans and found to be unacceptably immunogenic, theB-cell epitopes can be mapped and then altered to avoid immunedetection. Another approach uses methods to predict and remove potentialT-cell epitopes. Computational methods have been developed to scan andto identify the peptide sequences of biologic therapeutics with thepotential to bind to MHC proteins (Desmet et al. (2005) Proteins58:53-69. Alternatively a human dendritic cell-based method can be usedto identify CD4⁺ T-cell epitopes in potential protein allergens(Stickler et al. (2000) J. Immunother. 23: 654-60; Morrison and Schlom(1990) Important Adv. Oncol. 3-18; Riechmann et al. (1988) Nature332:323-327; Roguska et al. (1996) Protein Engineer. 9:895-904; Kashmiriet al. (2005) Methods (San Diego Calif.) 36(1):25-34; Desmet-Johan etal. 2005) Proteins 58:53-69; Stickler et al. (2000) J. Immunother.23:654-60.)

B 7. In Vivo Efficacy

To generate a DVD-Ig molecule with desired in vivo efficacy, it isimportant to generate and select mAbs with similarly desired in vivoefficacy when given in combination. However, in some instances theDVD-Ig may exhibit in vivo efficacy that cannot be achieved with thecombination of two separate mAbs. For instance, a DVD-Ig may bring twotargets in close proximity leading to an activity that cannot beachieved with the combination of two separate mAbs. Additional desirablebiological functions are described herein in section B 3. Parentantibodies with characteristics desirable in the DVD-Ig molecule may beselected based on factors such as pharmacokinetic t ½; tissuedistribution; soluble versus cell surface targets; and targetconcentration—soluble/density—surface.

B 8. In Vivo Tissue Distribution

To generate a DVD-Ig molecule with desired in vivo tissue distribution,in an embodiment parent mAbs with similar desired in vivo tissuedistribution profile must be selected. Alternatively, based on themechanism of the dual-specific targeting strategy, it may at other timesnot be required to select parent mAbs with the similarly desired in vivotissue distribution when given in combination. For instance, in the caseof a DVD-Ig in which one binding component targets the DVD-Ig to aspecific site thereby bringing the second binding component to the sametarget site. For example, one binding specificity of a DVD-Ig couldtarget pancreas (islet cells) and the other specificity could bring GLP1to the pancreas to induce insulin.

B 9. Isotype

To generate a DVD-Ig molecule with desired properties including, but notlimited to, isotype, effector functions and the circulating half-life,in an embodiment parent mAbs with appropriate Fc-effector functionsdepending on the therapeutic utility and the desired therapeuticend-point are selected. There are five main heavy-chain classes orisotypes some of which have several sub-types and these determine theeffector functions of an antibody molecule. These effector functionsreside in the hinge region, CH2 and CH3 domains of the antibodymolecule. However, residues in other parts of an antibody molecule mayhave effects on effector functions as well. The hinge region Fc-effectorfunctions include: (i) antibody-dependent cellular cytotoxicity, (ii)complement (C1q) binding, activation and complement-dependentcytotoxicity (CDC), (iii) phagocytosis/clearance of antigen-antibodycomplexes, and (iv) cytokine release in some instances. TheseFc-effector functions of an antibody molecule are mediated through theinteraction of the Fc-region with a set of class-specific cell surfacereceptors. Antibodies of the IgG1 isotype are most active while IgG2 andIgG4 having minimal or no effector functions. The effector functions ofthe IgG antibodies are mediated through interactions with threestructurally homologous cellular Fc receptor types (and sub-types)(FcgR1, FcgRII and FcgRIII). These effector functions of an IgG1 can beeliminated by mutating specific amino acid residues in the lower hingeregion (e.g., L234A, L235A) that are required for FcgR and C1q binding.Amino acid residues in the Fc region, in particular the CH2-CH3 domains,also determine the circulating half-life of the antibody molecule. ThisFc function is mediated through the binding of the Fc-region to theneonatal Fc receptor (FcRn) which is responsible for recycling ofantibody molecules from the acidic lysosomes back to the generalcirculation.

Whether a mAb should have an active or an inactive isotype will dependon the desired therapeutic end-point for an antibody. Some examples ofusage of isotypes and desired therapeutic outcome are listed below:

-   -   a) If the desired end-point is functional neutralization of a        soluble cytokine then an inactive isotype may be used;    -   b) If the desired out-come is clearance of a pathological        protein then an active isotype may be used;    -   c) If the desired out-come is clearance of protein aggregates        then an active isotype may be used;    -   d) If the desired outcome is to antagonize a surface receptor        then an inactive isotype is used (Tysabri, IgG4; OKT3, mutated        IgG1);    -   e) If the desired outcome is to eliminate target cells then an        active isotype is used (Herceptin, IgG1 (and with enhanced        effector functions); and    -   f) If the desired outcome is to clear proteins from circulation        without entering the CNS then an IgM isotype may be used (e.g.,        clearing circulating Ab peptide species).        The Fc effector functions of a parental mAb can be determined by        various in vitro methods well known in the art.

As discussed, the selection of isotype, and thereby the effectorfunctions will depend upon the desired therapeutic end-point. In caseswhere simple neutralization of a circulating target is desired, forexample blocking receptor-ligand interactions, the effector functionsmay not be required. In such instances isotypes or mutations in theFc-region of an antibody that eliminate effector functions aredesirable. In other instances where elimination of target cells is thetherapeutic end-point, for example elimination of tumor cells, isotypesor mutations or de-fucosylation in the Fc-region that enhance effectorfunctions are desirable (Presta (2006) Adv. Drug Delivery Rev.58:640-656; Satoh et al. (2006) Expert Opin. Biol. Ther. 6:1161-1173).Similarly, depending up on the therapeutic utility, the circulatinghalf-life of an antibody molecule can be reduced/prolonged by modulatingantibody-FcRn interactions by introducing specific mutations in the Fcregion (Dall'Acqua et al. (2006) J. Biol. Chem. 281:23514-23524; Petkovaet al. (2006) Internat. Immunol. 18:1759-1769; Vaccaro et al. (2007)Proc. Natl. Acad. Sci. USA 103:18709-18714).

The published information on the various residues that influence thedifferent effector functions of a normal therapeutic mAb may need to beconfirmed for DVD-Ig. It may be possible that in a DVD-Ig formatadditional (different) Fc-region residues, other than those identifiedfor the modulation of monoclonal antibody effector functions, may beimportant.

Overall, the decision as to which Fc-effector functions (isotype) willbe critical in the final DVD-Ig format will depend upon the diseaseindication, therapeutic target, desired therapeutic end-point and safetyconsiderations. Listed below are exemplary appropriate heavy chain andlight chain constant regions including, but not limited to:

-   -   IgG1—allotype: G1mz    -   IgG1 mutant—A234, A235    -   IgG2—allotype: G2m(n−)    -   Kappa—Km3    -   Lambda

Fc Receptor and C1q Studies:

The possibility of unwanted antibody-dependent cell-mediatedcytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC) byantibody complexing to any overexpressed target on cell membranes can beabrogated by the (for example, L234A, L235A) hinge-region mutations.These substituted amino acids, present in the IgG1 hinge region of mAb,are expected to result in diminished binding of mAb to human Fcreceptors (but not FcRn), as FcgR binding is thought to occur withinoverlapping sites on the IgG1 hinge region. This feature of mAb may leadto an improved safety profile over antibodies containing a wild-typeIgG. Binding of mAb to human Fc receptors can be determined by flowcytometry experiments using cell lines (e.g., THP-1, K562) and anengineered CHO cell line that expresses FcgRIIb (or other FcgRs).Compared to IgG1 control monoclonal antibodies, mAb show reduced bindingto FcgRI and FcgRIIa whereas binding to FcgRIIb is unaffected. Thebinding and activation of C1q by antigen/IgG immune complexes triggersthe classical complement cascade with consequent inflammatory and/orimmunoregulatory responses. The C1q binding site on IgGs has beenlocalized to residues within the IgG hinge region. C1q binding toincreasing concentrations of mAb was assessed by C1q ELISA. The resultsdemonstrate that mAb is unable to bind to C1q, as expected when comparedto the binding of a wildtype control IgG1. Overall, the L234A, L235Ahinge region mutation abolishes binding of mAb to FcgRI, FcgRIIa and C1qbut does not impact the interaction of mAb with FcgRIIb. These datasuggests that in vivo, mAb with mutant Fc will interact normally withthe inhibitory FcgRIIb but will likely fail to interact with theactivating FcgRI and FcgRIIa receptors or C1q.

Human FcRn binding:

The neonatal receptor (FcRn) is responsible for transport of IgG acrossthe placenta and to control the catabolic half-life of the IgGmolecules. It might be desirable to increase the terminal half-life ofan antibody to improve efficacy, to reduce the dose or frequency ofadministration, or to improve localization to the target. Alternatively,it might be advantageous to do the converse that is, to decrease theterminal half-life of an antibody to reduce whole body exposure or toimprove the target-to-non-target binding ratios. Tailoring theinteraction between IgG and its salvage receptor, FcRn, offers a way toincrease or decrease the terminal half-life of IgG. Proteins in thecirculation, including IgG, are taken up in the fluid phase throughmicropinocytosis by certain cells, such as those of the vascularendothelia. IgG can bind FcRn in endosomes under slightly acidicconditions (pH 6.0-6.5) and can recycle to the cell surface, where it isreleased under almost neutral conditions (pH 7.0-7.4). Mapping of theFc-region-binding site on FcRn80, 16, 17 showed that two histidineresidues that are conserved across species, His310 and His435, areresponsible for the pH dependence of this interaction. Usingphage-display technology, a mouse Fc-region mutation that increasesbinding to FcRn and extends the half-life of mouse IgG was identified(see Victor et al. (1997) Nature Biotechnol. 15(7):637-640). Fc-regionmutations that increase the binding affinity of human IgG for FcRn at pH6.0, but not at pH 7.4, have also been identified (see Dall'Acqua et al.(2002) J. Immunol. 169(9):5171-80). Moreover, in one case, a similarpH-dependent increase in binding (up to 27-fold) was also observed forrhesus FcRn, and this resulted in a twofold increase in serum half-lifein rhesus monkeys compared with the parent IgG (see Hinton et al. (2004)J. Biol. Chem. 279(8):6213-6216). These findings indicate that it isfeasible to extend the plasma half-life of antibody therapeutics bytailoring the interaction of the Fc region with FcRn. Conversely,Fc-region mutations that attenuate interaction with FcRn can reduceantibody half-life.

B.10 Pharmacokinetics (PK)

To generate a DVD-Ig molecule with desired pharmacokinetic profile, inan embodiment parent mAbs with the similarly desired pharmacokineticprofile are selected. One consideration is that immunogenic response tomonoclonal antibodies (i.e., HAHA, human anti-human antibody response;HACA, human anti-chimeric antibody response) further complicates thepharmacokinetics of these therapeutic agents. In an embodiment,monoclonal antibodies with minimal or no immunogenicity are used forconstructing DVD-Ig molecules such that the resulting DVD-Igs will alsohave minimal or no immunogenicity. Some of the factors that determinethe PK of a mAb include, but are not limited to, intrinsic properties ofthe mAb (VH amino acid sequence); immunogenicity; FcRn binding and Fcfunctions.

The PK profile of selected parental monoclonal antibodies can be easilydetermined in rodents as the PK profile in rodents correlates well with(or closely predicts) the PK profile of monoclonal antibodies incynomolgus monkey and humans. The PK profile is determined as describedin Example section 1.2.2.3.A.

After the parental monoclonal antibodies with desired PK characteristics(and other desired functional properties as discussed herein) areselected, the DVD-Ig is constructed. As the DVD-Ig molecules contain twoantigen-binding domains from two parental monoclonal antibodies, the PKproperties of the DVD-Ig are assessed as well. Therefore, whiledetermining the PK properties of the DVD-Ig, PK assays may be employedthat determine the PK profile based on functionality of bothantigen-binding domains derived from the 2 parent monoclonal antibodies.The PK profile of a DVD-Ig can be determined as described in Example1.2.2.3.A. Additional factors that may impact the PK profile of DVD-Iginclude the antigen-binding domain (CDR) orientation; linker size; andFc/FcRn interactions. PK characteristics of parent antibodies can beevaluated by assessing the following parameters: absorption,distribution, metabolism and excretion.

Absorption:

To date, administration of therapeutic monoclonal antibodies is viaparenteral routes (e.g., intravenous [IV], subcutaneous [SC], orintramuscular [IM]). Absorption of a mAb into the systemic circulationfollowing either SC or IM administration from the interstitial space isprimarily through the lymphatic pathway. Saturable, presystemic,proteolytic degradation may result in variable absolute bioavailabilityfollowing extravascular administration. Usually, increases in absolutebioavailability with increasing doses of monoclonal antibodies may beobserved due to saturated proteolytic capacity at higher doses. Theabsorption process for a mAb is usually quite slow as the lymph fluiddrains slowly into the vascular system, and the duration of absorptionmay occur over hours to several days. The absolute bioavailability ofmonoclonal antibodies following SC administration generally ranges from50% to 100%. In the case of a transport-mediating structure at theblood-brain barrier targeted by the DVD-Ig construct, circulation timesin plasma may be reduced due to enhanced trans-cellular transport at theblood brain barrier (BBB) into the CNS compartment, where the DVD-Ig isliberated to enable interaction via its second antigen recognition site.

Distribution:

Following IV administration, monoclonal antibodies usually follow abiphasic serum (or plasma) concentration-time profile, beginning with arapid distribution phase, followed by a slow elimination phase. Ingeneral, a biexponential pharmacokinetic model best describes this kindof pharmacokinetic profile. The volume of distribution in the centralcompartment (Vc) for a mAb is usually equal to or slightly larger thanthe plasma volume (2-3 liters). A distinct biphasic pattern in serum(plasma) concentration versus time profile may not be apparent withother parenteral routes of administration, such as IM or SC, because thedistribution phase of the serum (plasma) concentration-time curve ismasked by the long absorption portion. Many factors, includingphysicochemical properties, site-specific and target-oriented receptormediated uptake, binding capacity of tissue, and mAb dose can influencebiodistribution of a mAb. Some of these factors can contribute tononlinearity in biodistribution for a mAb.

Metabolism and Excretion:

Due to the molecular size, intact monoclonal antibodies are not excretedinto the urine via kidney. They are primarily inactivated by metabolism(e.g., catabolism). For IgG-based therapeutic monoclonal antibodies,half-lives typically ranges from hours or 1-2 days to over 20 days. Theelimination of a mAb can be affected by many factors, including, but notlimited to, affinity for the FcRn receptor, immunogenicity of the mAb,the degree of glycosylation of the mAb, the susceptibility for the mAbto proteolysis, and receptor-mediated elimination.

B.11 Tissue Cross-Reactivity Pattern on Human and Tox Species:

Identical staining pattern suggests that potential human toxicity can beevaluated in tox species. Tox species are those animal in whichunrelated toxicity is studied.

The individual antibodies are selected to meet two criteria. (1) tissuestaining appropriate for the known expression of the antibody target;and (2) similar staining pattern between human and tox species tissuesfrom the same organ.

Criterion 1: Immunizations and/or antibody selections typically employrecombinant or synthesized antigens (proteins, carbohydrates or othermolecules). Binding to the natural counterpart and counterscreen againstunrelated antigens are often part of the screening funnel fortherapeutic antibodies. However, screening against a multitude ofantigens is often unpractical. Therefore tissue cross-reactivity studieswith human tissues from all major organs serve to rule out unwantedbinding of the antibody to any unrelated antigens.

Criterion 2: Comparative tissue cross reactivity studies with human andtox species tissues (cynomolgus monkey, dog, possibly rodents andothers, the same 36 or 37 tissues are being tested as in the humanstudy) help to validate the selection of a tox species. In the typicaltissue cross-reactivity studies on frozen tissue sections therapeuticantibodies may demonstrate the expected binding to the known antigenand/or to a lesser degree binding to tissues based either on low levelinteractions (unspecific binding, low level binding to similar antigens,low level charge based interactions, etc.). In any case the mostrelevant toxicology animal species is the one with the highest degree ofcoincidence of binding to human and animal tissue.

Tissue cross reactivity studies follow the appropriate regulatoryguidelines including EC CPMP Guideline III/5271/94“Production andquality control of mAbs” and the 1997 US FDA/CBER “Points to Consider inthe Manufacture and Testing of Monoclonal Antibody Products for HumanUse”. Cryosections (5 μm) of human tissues obtained at autopsy or biopsywere fixed and dried on object glass. The peroxidase staining of tissuesections was performed, using the avidin-biotin system. FDA's Guidance“Points to Consider in the Manufacture and Testing of MonoclonalAntibody Products for Human Use”.

Tissue cross reactivity studies are often done in two stages, with thefirst stage including cryosections of 32 tissues (typically: AdrenalGland, Gastrointestinal Tract, Prostate, Bladder, Heart, SkeletalMuscle, Blood Cells, Kidney, Skin, Bone Marrow, Liver, Spinal Cord,Breast, Lung, Spleen, Cerebellum, Lymph Node, Testes, Cerebral Cortex,Ovary, Thymus, Colon, Pancreas, Thyroid, Endothelium, Parathyroid,Ureter, Eye, Pituitary, Uterus, Fallopian Tube and Placenta) from onehuman donor. In the second phase a full cross reactivity study isperformed with up to 38 tissues (including adrenal, blood, blood vessel,bone marrow, cerebellum, cerebrum, cervix, esophagus, eye, heart,kidney, large intestine, liver, lung, lymph node, breast mammary gland,ovary, oviduct, pancreas, parathyroid, peripheral nerve, pituitary,placenta, prostate, salivary gland, skin, small intestine, spinal cord,spleen, stomach, striated muscle, testis, thymus, thyroid, tonsil,ureter, urinary bladder, and uterus) from 3 unrelated adults. Studiesare done typically at minimally two dose levels.

The therapeutic antibody (i.e., test article) and isotype matchedcontrol antibody may be biotinylated for avidin-biotin complex (ABC)detection; other detection methods may include tertiary antibodydetection for a FITC (or otherwise) labeled test article, orprecomplexing with a labeled anti-human IgG for an unlabeled testarticle.

Briefly, cryosections (about 5 μm) of human tissues obtained at autopsyor biopsy are fixed and dried on object glass. The peroxidase stainingof tissue sections is performed, using the avidin-biotin system. First(in case of a precomplexing detection system), the test article isincubated with the secondary biotinylated anti-human IgG and developedinto immune complex. The immune complex at the final concentrations of 2and 10 μg/mL of test article is added onto tissue sections on objectglass and then the tissue sections are reacted for 30 minutes with aavidin-biotin-peroxidase kit. Subsequently, DAB (3,3′-diaminobenzidine),a substrate for the peroxidase reaction, is applied for 4 minutes fortissue staining. Antigen-Sepharose beads are used as positive controltissue sections.

Any specific staining is judged to be either an expected (e.g.,consistent with antigen expression) or unexpected reactivity based uponknown expression of the target antigen in question. Any staining judgedspecific is scored for intensity and frequency. Antigen or serumcompletion or blocking studies can assist further in determining whetherobserved staining is specific or nonspecific.

If two selected antibodies are found to meet the selectioncriteria—appropriate tissue staining, matching staining between humanand toxicology animal specific tissue—they can be selected for DVD-Iggeneration.

The tissue cross reactivity study has to be repeated with the finalDVD-Ig construct, but while these studies follow the same protocol asoutline herein, they are more complex to evaluate because any bindingcan come from any of the two parent antibodies, and any unexplainedbinding needs to be confirmed with complex antigen competition studies.

It is readily apparent that the complex undertaking of tissuecrossreactivity studies with a multispecific molecule like a DVD-Ig isgreatly simplified if the two parental antibodies are selected for (1)lack of unexpected tissue cross reactivity findings and (2) forappropriate similarity of tissue cross reactivity findings between thecorresponding human and toxicology animal species tissues.

B.12 Specificity and Selectivity:

To generate a DVD-Ig molecule with desired specificity and selectivity,one needs to generate and select parent mAbs with the similarly desiredspecificity and selectivity profile.

Binding studies for specificity and selectivity with a DVD-Ig can becomplex due to the four or more binding sites, two each for eachantigen. Briefly, binding studies using ELISA, BIAcore KinExA or otherinteraction studies with a DVD-Ig need to monitor the binding of one,two or more antigens to the DVD-Ig molecule. While BIAcore technologycan resolve the sequential, independent binding of multiple antigens,more traditional methods including ELISA or more modern techniques likeKinExA cannot. Therefore careful characterization of each parentantibody is critical. After each individual antibody has beencharacterized for specificity, confirmation of specificity retention ofthe individual binding sites in the DVD-Ig molecule is greatlysimplified.

It is readily apparent that the complex undertaking of determining thespecificity of a DVD-Ig is greatly simplified if the two parentalantibodies are selected for specificity prior to being combined into aDVD-Ig.

Antigen-antibody interaction studies can take many forms, including manyclassical protein interaction studies, including ELISA (Enzyme linkedimmunosorbent assay), Mass spectrometry, chemical cross linking, SECwith light scattering, equilibrium dialysis, gel permeation,ultrafiltration, gel chromatography, large-zone analytical SEC,micropreparative ultracentrifugation (sedimentation equilibrium),spectroscopic methods, titration microcalorimetry, sedimentationequilibrium (in analytical ultracentrifuge), sedimentation velocity (inanalytical centrifuge), surface plasmon resonance (including BIAcore).Relevant references include “Current Protocols in Protein Science”,Coligan et al. (eds.) Volume 3, chapters 19 and 20, published by JohnWiley & Sons Inc., and references included therein and “CurrentProtocols in Immunology”, Coligan et al. (eds.) published by John Wiley& Sons Inc and relevant references included therein.

Cytokine Release in Whole Blood: The interaction of mAb with human bloodcells can be investigated by a cytokine release assay (Wing (1995)Therapeut. Immunol. 2(4):183-190; “Current Protocols in Pharmacology”,et al. (eds.) published by John Wiley & Sons Inc; Madhusudan (2004)Clin. Canc. Res. 10(19):6528-6534; Cox (2006) J. Methods 38(4):274-282;Choi (2001) Eur. J. Immunol. 31(1):94-106). Briefly, variousconcentrations of mAb are incubated with human whole blood for 24 hours.The concentration tested should cover a wide range including finalconcentrations mimicking typical blood levels in patients (including butnot limited to 100 ng/ml-100 μg/ml). Following the incubation,supernatants and cell lysates were analyzed for the presence of IL-1Rα,TNF-α, IL-1b, IL-6 and IL-8. Cytokine concentration profiles generatedfor mAb were compared to profiles produced by a negative human IgGcontrol and a positive LPS or PHA control. The cytokine profiledisplayed by mAb from both cell supernatants and cell lysates wascomparable to control human IgG. In an embodiment, the monoclonalantibody does not interact with human blood cells to spontaneouslyrelease inflammatory cytokines.

Cytokine release studies for a DVD-Ig are complex due to the four ormore binding sites, two each for each antigen. Briefly, cytokine releasestudies as described herein measure the effect of the whole DVD-Igmolecule on whole blood or other cell systems, but can resolve whichportion of the molecule causes cytokine release. Once cytokine releasehas been detected, the purity of the DVD-Ig preparation has to beascertained, because some co-purifying cellular components can causecytokine release on their own. If purity is not the issue, fragmentationof DVD-Ig (including but not limited to removal of Fc portion,separation of binding sites, etc.), binding site mutagenesis or othermethods may need to be employed to deconvolute any observations. It isreadily apparent that this complex undertaking is greatly simplified ifthe two parental antibodies are selected for lack of cytokine releaseprior to being combined into a DVD-Ig.

B.13 Cross Reactivity to Other Species for Toxicological Studies:

In an embodiment, the individual antibodies that are selected havesufficient cross-reactivity to appropriate tox species, for example,cynomolgus monkey. Parental antibodies need to bind to orthologousspecies target (i.e., cynomolgus monkey) and elicit appropriate response(modulation, neutralization, activation). In an embodiment, thecross-reactivity (affinity/potency) to orthologous species target shouldbe within 10-fold of the human target. In practice, the parentalantibodies are evaluated for multiple species, including mouse, rat,dog, monkey (and other non-human primates), as well as disease modelspecies (i.e., sheep for asthma model). The acceptable cross-reactivityto tox species from the parental monoclonal antibodies allows futuretoxicology studies of DVD-Ig-Ig in the same species. For that reason,the two parental monoclonal antibodies should have acceptablecross-reactivity for a common tox species therefore allowing toxicologystudies of DVD-Ig in the same species.

Parent mAbs may be selected from various mAbs that bind specific targetsand are well known in the art. These include, but are not limited toanti-TNF antibody (U.S. Pat. No. 6,258,562), anti-IL-12 and/oranti-IL-12p40 antibody (U.S. Pat. No. 6,914,128); anti-IL-18 antibody(US Patent Application No. 20050147610), anti-C5, anti-CBL, anti-CD147,anti-gp120, anti-VLA-4, anti-CD11a, anti-CD18, anti-VEGF, anti-CD40L,anti CD-40 (e.g., see PCT Publication No. WO2007124299) anti-Id,anti-ICAM-1, anti-CXCL13, anti-CD2, anti-EGFR, anti-TGF-beta 2,anti-HGF, anti-cMet, anti DLL-4, anti-NPR1, anti-PLGF, anti-ErbB3,anti-E-selectin, anti-Fact VII, anti-Her2/neu, anti-F gp, anti-CD11/18,anti-CD14, anti-ICAM-3, anti-RON, anti CD-19, anti-CD80 (e.g., see PCTPublication No. WO2003039486, anti-CD4, anti-CD3, anti-CD23,anti-beta2-integrin, anti-alpha4beta7, anti-CD52, anti-HLA DR, anti-CD22(e.g., see U.S. Pat. No. 5,789,554), anti-CD20, anti-MIF, anti-CD64(FcR), anti-TCR alpha beta, anti-CD2, anti-Hep B, anti-CA 125,anti-EpCAM, anti-gp120, anti-CMV, anti-gpIIbIIIa, anti-IgE, anti-CD25,anti-CD33, anti-HLA, anti-IGF1,2, anti IGFR, anti-VNRintegrin,anti-IL-1alpha, anti-IL-1beta, anti-IL-1 receptor, anti-IL-2 receptor,anti-IL-4, anti-IL-4 receptor, anti-IL5, anti-IL-5 receptor, anti-IL-6,anti-IL-6R, RANKL, NGF, DKK, alphaVbeta3, IL-17A, anti-IL-8, anti-IL-9,anti-IL-13, anti-IL-13 receptor, anti-IL-17, and anti-IL-23; IL-23p19;(see Presta (2005) J. Allergy Clin. Immunol. 116:731-6.

Parent mAbs may also be selected from various therapeutic antibodiesapproved for use, in clinical trials, or in development for clinicaluse. Such therapeutic antibodies include, but are not limited to,rituximab (Rituxan®, IDEC/Genentech/Roche) (see for example U.S. Pat.No. 5,736,137), a chimeric anti-CD20 antibody approved to treatNon-Hodgkin's lymphoma; HuMax-CD20, an anti-CD20 currently beingdeveloped by Genmab, an anti-CD20 antibody described in U.S. Pat. No.5,500,362, AME-133 (Applied Molecular Evolution), hA20 (Immunomedics,Inc.), HumaLYM (Intracel), and PRO70769 (PCT Application No.PCT/US2003/040426), trastuzumab (Herceptin®, Genentech) (see for exampleU.S. Pat. No. 5,677,171), a humanized anti-Her2/neu antibody approved totreat breast cancer; pertuzumab (rhuMab-2C4, Omnitarg®), currently beingdeveloped by Genentech; an anti-Her2 antibody described in U.S. Pat. No.4,753,894; cetuximab (Erbitux®, Imclone) (U.S. Pat. No. 4,943,533; PCTPublication No. WO 96/40210), a chimeric anti-EGFR antibody in clinicaltrials for a variety of cancers; ABX-EGF (U.S. Pat. No. 6,235,883),currently being developed by Abgenix-Immunex-Amgen; HuMaxEGFr (U.S. Pat.No. 7,247,301), currently being developed by Genmab; 425, EMD55900,EMD62000, and EMD72000 (Merck KGaA) (U.S. Pat. No. 5,558,864; Murthy etal. (1987) Arch. Biochem. Biophys. 252(2):549-60; Rodeck et al. (1987)J. Cell. Biochem. 35(4):315-20; Kettleborough et al. (1991) Protein Eng.4(7):773-83); ICR62 (Institute of Cancer Research) (PCT Publication No.WO 95/20045; Modjtahedi et al. (1993) J. Cell Biophys. 22(1-3):129-46;Modjtahedi et al. (1993) Br. J. Cancer 67(2):247-53; Modjtahedi et al.(1996) Br. J. Cancer 73(2):228-35; Modjtahedi et al. (2003) Int. J.Cancer 105(2):273-80); TheraCIM hR3 (YM Biosciences, Canada and Centrode Immunologia Molecular, Cuba (U.S. Pat. No. 5,891,996; U.S. Pat. No.6,506,883; Mateo et al. (1997) Immunotechnol. 3(1):71-81); mAb-806(Ludwig Institute for Cancer Research, Memorial Sloan-Kettering)(Jungbluth et al. (2003) Proc. Natl. Acad. Sci. USA 100(2):639-44);KSB-102 (KS Biomedix); MR1-1 (IVAX, National Cancer Institute) (PCTPublication No. WO 0162931); and SC100 (Scancell) (PCT WO 01/88138);alemtuzumab (Campath®, Millenium), a humanized mAb currently approvedfor treatment of B-cell chronic lymphocytic leukemia; muromonab-CD3(Orthoclone OKT3®), an anti-CD3 antibody developed by OrthoBiotech/Johnson & Johnson, ibritumomab tiuxetan (Zevalin®), an anti-CD20antibody developed by IDEC/Schering AG, gemtuzumab ozogamicin(Mylotarg®), an anti-CD33 (p67 protein) antibody developed byCelltech/Wyeth, alefacept (Amevive®), an anti-LFA-3 Fc fusion developedby Biogen), abciximab (ReoPro®), developed by Centocor/Lilly,basiliximab (Simulect®), developed by Novartis, palivizumab (Synagis®),developed by Medimmune, infliximab (Remicade®), an anti-TNFalphaantibody developed by Centocor, adalimumab (Humira®), an anti-TNFalphaantibody developed by Abbott, Humicade®, an anti-TNFalpha antibodydeveloped by Celltech, golimumab (CNTO-148), a fully human TNF antibodydeveloped by Centocor, etanercept (Enbrel®), an p75 TNF receptor Fcfusion developed by Immunex/Amgen, lenercept, an p55TNF receptor Fcfusion previously developed by Roche, ABX-CBL, an anti-CD147 antibodybeing developed by Abgenix, ABX-IL8, an anti-IL8 antibody beingdeveloped by Abgenix, ABX-MA1, an anti-MUC18 antibody being developed byAbgenix, Pemtumomab (R1549, 90Y-muHMFG1), an anti-MUC1 in development byAntisoma, Therex (R1550), an anti-MUC1 antibody being developed byAntisoma, AngioMab (AS1405), being developed by Antisoma, HuBC-1, beingdeveloped by Antisoma, Thioplatin (AS1407) being developed by Antisoma,Antegren® (natalizumab), an anti-alpha-4-beta-1 (VLA-4) andalpha-4-beta-7 antibody being developed by Biogen, VLA-1 mAb, ananti-VLA-1 integrin antibody being developed by Biogen, LTBR mAb, ananti-lymphotoxin beta receptor (LTBR) antibody being developed byBiogen, CAT-152, an anti-TGF-β antibody being developed by CambridgeAntibody Technology, ABT 874 (J695), an anti-IL-12 p40 antibody beingdeveloped by Abbott, CAT-192, an anti-TGFβ1 antibody being developed byCambridge Antibody Technology and Genzyme, CAT-213, an anti-Eotaxin1antibody being developed by Cambridge Antibody Technology, LymphoStat-B®an anti-Blys antibody being developed by Cambridge Antibody Technologyand Human Genome Sciences Inc., TRAIL-R1 mAb, an anti-TRAIL-R1 antibodybeing developed by Cambridge Antibody Technology and Human GenomeSciences, Inc., Avastin® bevacizumab, rhuMAb-VEGF), an anti-VEGFantibody being developed by Genentech, an anti-HER receptor familyantibody being developed by Genentech, Anti-Tissue Factor (ATF), ananti-Tissue Factor antibody being developed by Genentech, Xolair®(Omalizumab), an anti-IgE antibody being developed by Genentech,Raptiva® (Efalizumab), an anti-CD11a antibody being developed byGenentech and Xoma, MLN-02 Antibody (formerly LDP-02), being developedby Genentech and Millenium Pharmaceuticals, HuMax CD4, an anti-CD4antibody being developed by Genmab, HuMax-IL15, an anti-IL15 antibodybeing developed by Genmab and Amgen, HuMax-Inflam, being developed byGenmab and Medarex, HuMax-Cancer, an anti-Heparanase I antibody beingdeveloped by Genmab and Medarex and Oxford GcoSciences, HuMax-Lymphoma,being developed by Genmab and Amgen, HuMax-TAC, being developed byGenmab, IDEC-131, and anti-CD40L antibody being developed by IDECPharmaceuticals, IDEC-151 (Clenoliximab), an anti-CD4 antibody beingdeveloped by IDEC Pharmaceuticals, IDEC-114, an anti-CD80 antibody beingdeveloped by IDEC Pharmaceuticals, IDEC-152, an anti-CD23 beingdeveloped by IDEC Pharmaceuticals, anti-macrophage migration factor(MIF) antibodies being developed by IDEC Pharmaceuticals, BEC2, ananti-idiotypic antibody being developed by Imclone, IMC-1C11, ananti-KDR antibody being developed by Imclone, DC101, an anti-flk-1antibody being developed by Imclone, anti-VE cadherin antibodies beingdeveloped by Imclone, CEA-Cide® (labetuzumab), an anti-carcinoembryonicantigen (CEA) antibody being developed by Immunomedics, LymphoCide®(Epratuzumab), an anti-CD22 antibody being developed by Immunomedics,AFP-Cide, being developed by Immunomedics, MyelomaCide, being developedby Immunomedics, LkoCide, being developed by Immunomedics, ProstaCide,being developed by Immunomedics, MDX-010, an anti-CTLA4 antibody beingdeveloped by Medarex, MDX-060, an anti-CD30 antibody being developed byMedarex, MDX-070 being developed by Medarex, MDX-018 being developed byMedarex, Osidem® (IDM-1), and anti-Her2 antibody being developed byMedarex and Immuno-Designed Molecules, HuMax®-CD4, an anti-CD4 antibodybeing developed by Medarex and Genmab, HuMax-IL15, an anti-IL15 antibodybeing developed by Medarex and Genmab, CNTO 148, an anti-TNFα antibodybeing developed by Medarex and Centocor/J&J, CNTO 1275, an anti-cytokineantibody being developed by Centocor/J&J, MOR101 and MOR102,anti-intercellular adhesion molecule-1 (ICAM-1) (CD54) antibodies beingdeveloped by MorphoSys, MOR201, an anti-fibroblast growth factorreceptor 3 (FGFR-3) antibody being developed by MorphoSys, Nuvion®(visilizumab), an anti-CD3 antibody being developed by Protein DesignLabs, HuZAF®, an anti-gamma interferon antibody being developed byProtein Design Labs, Anti-α 5β1 Integrin, being developed by ProteinDesign Labs, anti-IL-12, being developed by Protein Design Labs, ING-1,an anti-Ep-CAM antibody being developed by Xoma, Xolair® (Omalizumab) ahumanized anti-IgE antibody developed by Genentech and Novartis, andMLN01, an anti-Beta2 integrin antibody being developed by Xoma. Inanother embodiment, the therapeutics include KRN330 (Kirin); huA33antibody (A33, Ludwig Institute for Cancer Research); CNTO 95 (alpha Vintegrins, Centocor); MEDI-522 (alpha Vβ3 integrin, Medimmune);volociximab (alpha Vfβ1 integrin, Biogen/PDL); Human mAb 216 (B cellglycosolated epitope, NCl); BiTE MT103 (bispecific CD19×CD3, Medimmune);4G7×H22 (Bispecific BcellxFcgammaR1, Medarex/Merck KGa); rM28(Bispecific CD28×MAPG, EP Patent No. EP1444268); MDX447 (EMD 82633)(Bispecific CD64×EGFR, Medarex); Catumaxomab (removab) (Bispecific EpCAMx anti-CD3, Trion/Fres); Ertumaxomab (bispecific HER2/CD3, FreseniusBiotech); oregovomab (OvaRex) (CA-125, ViRexx); Rencarex® (WX G250)(carbonic anhydrase IX, Wilex); CNTO 888 (CCL2, Centocor); TRC105 (CD105(endoglin), Tracon); BMS-663513 (CD137 agonist, Brystol Myers Squibb);MDX-1342 (CD19, Medarex); Siplizumab (MEDI-507) (CD2, Medimmune);Ofatumumab (Humax-CD20) (CD20, Genmab); Rituximab (Rituxan) (CD20,Genentech); veltuzumab (hA20) (CD20, Immunomedics); Epratuzumab (CD22,Amgen); lumiliximab (IDEC 152) (CD23, Biogen); muromonab-CD3 (CD3,Ortho); HuM291 (CD3 fc receptor, PDL Biopharma); HeFi-1, CD30, NCl);MDX-060 (CD30, Medarex); MDX-1401 (CD30, Medarex); SGN-30 (CD30, SeattleGenentics); SGN-33 (Lintuzumab) (CD33, Seattle Genentics); Zanolimumab(HuMax-CD4) (CD4, Genmab); HCD122 (CD40, Novartis); SGN-40 (CD40,Seattle Genentics); Campathlh (Alemtuzumab) (CD52, Genzyme); MDX-1411(CD70, Medarex); hLL1 (EPB-1) (CD74.38, Immunomedics); Galiximab(IDEC-144) (CD80, Biogen); MT293 (TRC093/D93) (cleaved collagen,Tracon); HuLuc63 (CS1, PDL Pharma); ipilimumab (MDX-010) (CTLA4, BrystolMyers Squibb); Tremelimumab (Ticilimumab, CP-675,2) (CTLA4, Pfizer);HGS-ETR1 (Mapatumumab) (DR4TRAIL-R1 agonist, Human Genome Science/GlaxoSmith Kline); AMG-655 (DR5, Amgen); Apomab (DR5, Genentech); CS-1008(DR5, Daiichi Sankyo); HGS-ETR2 (lexatumumab) (DR5TRAIL-R2 agonist,HGS); Cetuximab (Erbitux) (EGFR, Imclone); IMC-11F8, (EGFR, Imclone);Nimotuzumab (EGFR, YM Bio); Panitumumab (Vectabix) (EGFR, Amgen);Zalutumumab (HuMaxEGFr) (EGFR, Genmab); CDX-110 (EGFRvIII, AVANTImmunotherapeutics); adecatumumab (MT201) (Epcam , Merck); edrecolomab(Panorex, 17-1A) (Epcam , Glaxo/Centocor); MORAb-003 (folate receptor a,Morphotech); KW-2871 (ganglioside GD3, Kyowa); MORAb-009 (GP-9,Morphotech); CDX-1307 (MDX-1307) (hCGb, Celldex); Trastuzumab(Herceptin) (HER2, Celldex); Pertuzumab (rhuMAb 2C4) (HER2 (DI),Genentech); apolizumab (HLA-DR beta chain, PDL Pharma); AMG-479 (IGF-1R,Amgen); anti-IGF-1R R1507 (IGF1-R, Roche); CP 751871 (IGF1-R, Pfizer);IMC-A12 (IGF1-R, Imclone); BIIB022 (IGF-1R, Biogen); Mik-beta-1 (IL-2Rb(CD122), Hoffinan LaRoche); CNTO 328 (IL6, Centocor); Anti-KIR (1-7F9)(Killer cell Ig-like Receptor (KIR), Novo); Hu35193 (Lewis (y), Wyeth,Ludwig Institute of Cancer Research); hCBE-11 (LTβR, Biogen); HuHMFG1(MUC1, Antisoma/NCl); RAV12 (N-linked carbohydrate epitope, Raven); CAL(parathyroid hormone-related protein (PTH-rP), University ofCalifornia); CT-011 (PD1, CureTech); MDX-1106 (ono-4538) (PD1,Medarex/Ono); MAb CT-011 (PD1, Curetech); IMC-3G3 (PDGFRa, Imclone);bavituximab (phosphatidylserine, Peregrine); huJ591 (PSMA, CornellResearch Foundation); muJ591 (PSMA, Cornell Research Foundation); GC1008(TGFb (pan) inhibitor (IgG4), Genzyme); Infliximab (Remicade) (TNFa,Centocor); A27.15 (transferrin receptor, Salk Institute, INSERN WO2005/111082); E2.3 (transferrin receptor, Salk Institute); Bevacizumab(Avastin) (VEGF, Genentech); HuMV833 (VEGF, Tsukuba Research Lab) PCTPublication No. WO/2000/034337, University of Texas); IMC-18F1 (VEGFR1,Imclone); IMC-1121 (VEGFR2, Imclone).

B. Construction of DVD Molecules:

The dual variable domain immunoglobulin (DVD-Ig) molecule is designedsuch that two different light chain variable domains (VL) from the twodifferent parent monoclonal antibodies are linked in tandem directly orvia a short linker by recombinant DNA techniques, followed by the lightchain constant domain and, optionally, an Fc region. Similarly, theheavy chain comprises two different heavy chain variable domains (VH)linked in tandem, followed by the constant domain CH1 and Fc region(FIG. 1A).

The variable domains can be obtained using recombinant DNA techniquesfrom a parent antibody generated by any one of the methods describedherein. In an embodiment, the variable domain is a murine heavy or lightchain variable domain. In another embodiment, the variable domain is aCDR grafted or a humanized variable heavy or light chain domain. In anembodiment, the variable domain is a human heavy or light chain variabledomain.

In one embodiment the first and second variable domains are linkeddirectly to each other using recombinant DNA techniques. In anotherembodiment the variable domains are linked via a linker sequence. In anembodiment, two variable domains are linked. Three or more variabledomains may also be linked directly or via a linker sequence. Thevariable domains may bind the same antigen or may bind differentantigens. DVD molecules of the invention may include one immunoglobulinvariable domain and one non-immunoglobulin variable domain such asligand binding domain of a receptor, active domain of an enzyme. DVDmolecules may also comprise 2 or more non-Ig domains.

The linker sequence may be a single amino acid or a polypeptidesequence. In an embodiment, the linker sequences are selected from thegroup consisting of AKTTPKLEEGEFSEAR (SEQ ID NO: 1); AKTTPKLEEGEFSEARV(SEQ ID NO: 2); AKTTPKLGG (SEQ ID NO: 3); SAKTTPKLGG (SEQ ID NO: 4);SAKTTP (SEQ ID NO: 5); RADAAP (SEQ ID NO: 6); RADAAPTVS (SEQ ID NO: 7);RADAAAAGGPGS (SEQ ID NO: 8); RADAAAA(G₄S)₄ (SEQ ID NO: 9);SAKTTPKLEEGEFSEARV (SEQ ID NO: 10); ADAAP (SEQ ID NO: 11); ADAAPTVSIFPP(SEQ ID NO: 12); TVAAP (SEQ ID NO: 13); TVAAPSVFIFPP (SEQ ID NO: 14);QPKAAP (SEQ ID NO: 15); QPKAAPSVTLFPP (SEQ ID NO: 16); AKTTPP (SEQ IDNO: 17); AKTTPPSVTPLAP (SEQ ID NO: 18); AKTTAP (SEQ ID NO: 19);AKTTAPSVYPLAP (SEQ ID NO: 20); ASTKGP (SEQ ID NO: 21); ASTKGPSVFPLAP(SEQ ID NO: 22), GGGGSGGGGSGGGGS (SEQ ID NO: 23); GENKVEYAPALMALS (SEQID NO: 24); GPAKELTPLKEAKVS (SEQ ID NO: 25); GHEAAAVMQVQYPAS (SEQ ID NO:26), TVAAPSVFIFPPTVAAPSVFIFPP (SEQ ID NO: 27); andASTKGPSVFPLAPASTKGPSVFPLAP (SEQ ID NO: 28). The choice of linkersequences is based on crystal structure analysis of several Fabmolecules. There is a natural flexible linkage between the variabledomain and the CH1/CL constant domain in Fab or antibody molecularstructure. This natural linkage comprises approximately 10-12 amino acidresidues, contributed by 4-6 residues from C-terminus of V domain and4-6 residues from the N-terminus of CL/CH1 domain. DVD Igs of theinvention were generated using N-terminal 5-6 amino acid residues, or11-12 amino acid residues, of CL or CH1 as linker in light chain andheavy chain of DVD-Ig, respectively. The N-terminal residues of CL orCH1 domains, particularly the first 5-6 amino acid residues, adopt aloop conformation without strong secondary structures, therefore can actas flexible linkers between the two variable domains. The N-terminalresidues of CL or CH1 domains are natural extension of the variabledomains, as they are part of the Ig sequences, therefore minimize to alarge extent any immunogenicity potentially arising from the linkers andjunctions.

Other linker sequences may include any sequence of any length of CL/CH1domain but not all residues of CL/CH1 domain; for example the first 5-12amino acid residues of the CL/CH1 domains; the light chain linkers canbe from Cκ or Cλ; and the heavy chain linkers can be derived from CH1 ofany isotypes, including Cγ1, Cγ2, Cγ3, Cγ4, Cα1, Cα2, Cδ, Cε, and Cμ.Linker sequences may also be derived from other proteins such as Ig-likeproteins, (e.g.,TCR, FcR, KIR); G/S based sequences (e.g., G4S repeatsSEQ ID NO: 29); hinge region-derived sequences; and other naturalsequences from other proteins.

In an embodiment a constant domain is linked to the two linked variabledomains using recombinant DNA techniques. In an embodiment, sequencecomprising linked heavy chain variable domains is linked to a heavychain constant domain and sequence comprising linked light chainvariable domains is linked to a light chain constant domain. In anembodiment, the constant domains are human heavy chain constant domainand human light chain constant domain respectively. In an embodiment,the DVD heavy chain is further linked to an Fc region. The Fc region maybe a native sequence Fc region, or a variant Fc region. In anotherembodiment, the Fc region is a human Fc region. In another embodimentthe Fc region includes Fc region from IgG1, IgG2, IgG3, IgG4, IgA, IgM,IgE, or IgD.

In another embodiment two heavy chain DVD polypeptides and two lightchain DVD polypeptides are combined to form a DVD-Ig molecule. Table 2lists amino acid sequences of VH and VL regions of exemplary antibodiesfor targets useful for treating disease, e.g., for treating cancer. Inan embodiment, the invention provides a DVD comprising at least two ofthe VH and/or VL regions listed in Table 2, in any orientation.

TABLE 2 List of Amino Acid Sequences of VH and VL regions ofAntibodies for Generating DVD-12s SEQ Protein Sequence ID NO Unique IDRegion 1234567890123456789012345678901234567890 30 E26.13VH VH 1L-βEVQLVESGGGVVQPGRSLRLSCSASGFIFSRYDMSWVRQA (seq. 1)PGKGLEWVAYISHGGAGTYYPDSVKGRFTISRDNSKNTLFLQMDSLRPEDTGVYFCARGGVTKGYFDVWGQGTPVTVSS 31 E26.13VL VL 1L-βDIQMTQSPSSLSASVGDRVTITCRASGNIHNYLTWYQQTP (seq. 1)GKAPKLLIYNAKTLADGVPSRFSGSGSGTDYTFTISSLQP EDIATYYCQHFWSIPYTFGQGTKLQITR 32E26.35VH VH 1L-β EVQLVESGGGVVQPGRSLRLSCSASGFIFSRYDMSWVRQA (seq. 2)PGKGLEWVAYISHGGAGTYYPDSVKGRFTISRDNSKNTLFLQMDSLRAEDTAVYYCARGGVYKGYFDVWGQGTPVTVSS 33 E26.35VL VL 1L-βDIQMTQSPSSLSASVGDRVTITCRASGNIHNYLTWYQQTP (seq. 2)GKAPKLLIYNAKTLADGVPSRFSGSGSGTDYTFTISSLQP EDIATYYCQHFWSIPYTFGQGTKLQITR 341B12.1VH VH 1L-β EVQLQESGPGLVKPSETLSLTCTVSGFSLSDYGVSWIRQP (seq. 3)PGKGLEWLGLIWGGGDTYYNSPLKSRLTISKDNSKSQVSLKLSSVTAADTAVYYCAKQRTLWGYDLYGMDYWGQGTLVTV SS 35 1B12.1VL VL 1L-βDTQVTQSPSSLSASVGDRVTITCITSTDIDVDMNWYQQKP (seq. 3)GKPPKLLISQGNTLRPGVPSRFSSSGSGTDFTFTISSLQP EDFATYYCLQSDNLPLTFGQGTKLEIKR 361B12.3VH VH 1L-β EVQLQESGPGLVKPSETLSLTCTVSGFSLSDYGVSWIRQP (seq. 4)PGKGLEWLGLIWGGGDTYYNSPLKSRLTISKDNSKSQVSLKLSSVTAADTAVYYCAKQRTLWGYDLYGMDYWGQGTLVTV SS 37 1B12.3VL VL 1L-βDTVVTQSPAFLSVTPGEKVTITCITSTDIDVDMNWYQQKP (seq. 4)DQPPKLLISQGNTLRPGVPSRFSSSGSGTDFTFTISSLEA EDAATYYCLQSDNLPLTFGQGTKLEIKR 381B12.6VH VH 1L-β EVQLVESGGGLVQPGGSLRLSCAVSGFTLSDYGVSWIRQA (seq. 5)PGKGLEWLGLIWGGGDTYYNSPLKSRLTISKDNSKSTVYLQMNSLRAEDTAVYYCAKQRTLWGYDLYGMDYWGQGTLVTV SS 39 1B12.6VL VL 1L-βETTVTQSPSSLSASVGDRVTITCITSTDIDVDMNWYQQKP (seq. 5)GKPPKLLISQGNTLRPGVPSRFSSSGSGTDFTFTISSLQP EDFATYYCLQSDNLPLTFGQGTKLEIKR 403D12 VH 1L-α EVQLVQSGAEVKKPGVSVKVSCKASGYTFTTYGMHWVRQA 10/15VH (seq. 3)PGQGLEWMGWINTYTGESTYADDFQGRVTFTLDTSTSTAYMELSSLRSEDTAVYFCARGIYYYGSSYAMNYWGQGTTVTV SS 41 3D12 VL 1L-αDIQMTQSPSSLSASVGDRVTITCRASQDISNMLNWYQQKP 10/15VL (seq. 3)GKTPKLLIYYTSRLYPGVPSRFSGSGSGTDYTFTISSLQP EDIATYFCQQGKTLPYAFGQGTKLEIKR 423D12.16VH VH 1L-α EIQLVQSGAEVKKPGASVKVSCKASGYTFTNYGMNWVRQA (seq. 2)PGQDLERMAWINTYTGESTYADDFKGRFTFTLDTSTSTAYMELSSLRSEDTAVYFCARGIYYYGSSYAMDYWGQGTTVTV SS 43 3D12.16VL VL 1L-αDIQMTQSPSSLSASVGDRVTITCRASQDISNCLNWYQQKP (seq. 2)GKTPKLLIYYTSRLHSGVPSRFSGSGSGTDYTFTISSLQP EDIATYFCQQGKTLPYAFGQGTKLEIKR 443D12.8VH VH 1L-α EIQLVQSGSELKKPGASVKVSCKASGYTFTNYGMNWVRQA (seq.1)PGQDLERMAWINTYTGESTYADDFKGRFVFSLDTSVSTAYLQISSLKAEDTAVYFCARGIYYYGSSYAMDYWGQGTTVTV SS 45 3D12.8VL VL 1L-αDIQMTQSPSSLSASVGDRVTITCRASQDISNCLNWYQQKP (seq.1)GKTPKLLIYYTSRLHSGVPSRFSGSGSGTDYTFTISSLQP EDIATYFCQQGKTLPYAFGQGTKLEIKR 463D12r16VH VH 1L-α EVQLVQSGAEVKKPGASVKVSCKASGYTFKYYGMNWVRQA (seq.4)PGQGLERMGWINTYTGQSTYADDFKGRVTFTLDTSTSTAYMELSSLRSEDTAVYYCARDIYYYGSDFAMDYWGQGTTVTV SS 47 3D12r16VL VL 1L-αDIQMTQSPSSLSASVGDRVTITCRASQDISNMLNWYQQKP (seq.4)GKAPKLLIYYTSRLKPGVPSRFSGSGSGTDYTFTISSLQP EDIATYFCQQGKTAPYTFGQGTKLEIKR

Detailed description of specific DVD-Ig molecules that bind specifictargets, and methods of making the same, is provided in the Examplessection below.

C. Production of DVD Proteins

Binding proteins of the present invention may be produced by any of anumber of techniques known in the art. For example, expression from hostcells, wherein expression vector(s) encoding the DVD heavy and DVD lightchains is (are) transfected into a host cell by standard techniques. Thevarious forms of the term “transfection” are intended to encompass awide variety of techniques commonly used for the introduction ofexogenous DNA into a prokaryotic or eukaryotic host cell, e.g.,electroporation, calcium-phosphate precipitation, DEAE-dextrantransfection and the like. Although it is possible to express the DVDproteins of the invention in either prokaryotic or eukaryotic hostcells, DVD proteins are expressed in eukaryotic cells, for example,mammalian host cells, because such eukaryotic cells (and in particularmammalian cells) are more likely than prokaryotic cells to assemble andsecrete a properly folded and immunologically active DVD protein.

Exemplary mammalian host cells for expressing the recombinant antibodiesof the invention include Chinese Hamster Ovary (CHO cells) (includingdhfr-CHO cells, described in Urlaub and Chasin (1980) Proc. Natl. Acad.Sci. USA 77:4216-4220, used with a DHFR selectable marker, e.g., asdescribed in Kaufman and Sharp (1982) Mol. Biol. 159:601-621), NS0myeloma cells, COS cells, SP2 and PER.C6 cells. When recombinantexpression vectors encoding DVD proteins are introduced into mammalianhost cells, the DVD proteins are produced by culturing the host cellsfor a period of time sufficient to allow for expression of the DVDproteins in the host cells or secretion of the DVD proteins into theculture medium in which the host cells are grown. DVD proteins can berecovered from the culture medium using standard protein purificationmethods.

In an exemplary system for recombinant expression of DVD proteins of theinvention, a recombinant expression vector encoding both the DVD heavychain and the DVD light chain is introduced into dhfr-CHO cells bycalcium phosphate-mediated transfection. Within the recombinantexpression vector, the DVD heavy and light chain genes are eachoperatively linked to CMV enhancer/AdMLP promoter regulatory elements todrive high levels of transcription of the genes. The recombinantexpression vector also carries a DHFR gene, which allows for selectionof CHO cells that have been transfected with the vector usingmethotrexate selection/amplification. The selected transformant hostcells are cultured to allow for expression of the DVD heavy and lightchains and intact DVD protein is recovered from the culture medium.Standard molecular biology techniques are used to prepare therecombinant expression vector, transfect the host cells, select fortransformants, culture the host cells and recover the DVD protein fromthe culture medium. Still further the invention provides a method ofsynthesizing a DVD protein of the invention by culturing a host cell ofthe invention in a suitable culture medium until a DVD protein of theinvention is synthesized. The method can further comprise isolating theDVD protein from the culture medium.

An important feature of DVD-Ig is that it can be produced and purifiedin a similar way as a conventional antibody. The production of DVD-Igresults in a homogeneous, single major product with desireddual-specific activity, without any sequence modification of theconstant region or chemical modifications of any kind. Other previouslydescribed methods to generate “bi-specific”, “multi-specific”, and“multi-specific multivalent” full length binding proteins do not lead toa single primary product but instead lead to the intracellular orsecreted production of a mixture of assembled inactive, mono-specific,multi-specific, multivalent, full length binding proteins, andmultivalent full length binding proteins with combination of differentbinding sites. As an example, based on the design described by PCTPublication WO2001/077342, there are 16 possible combinations of heavyand light chains. Consequently only 6.25% of protein is likely to be inthe desired active form, and not as a single major product or singleprimary product compared to the other 15 possible combinations.Separation of the desired, fully active forms of the protein frominactive and partially active forms of the protein using standardchromatography techniques, typically used in large scale manufacturing,is yet to be demonstrated.

Surprisingly the design of the “dual-specific multivalent full lengthbinding proteins” of the present invention leads to a dual variabledomain light chain and a dual variable domain heavy chain which assembleprimarily to the desired “dual-specific multivalent full length bindingproteins”.

At least 50%, at least 75% and at least 90% of the assembled, andexpressed dual variable domain immunoglobulin molecules are the desireddual-specific tetravalent protein. This aspect of the inventionparticularly enhances the commercial utility of the invention.Therefore, the present invention includes a method to express a dualvariable domain light chain and a dual variable domain heavy chain in asingle cell leading to a single primary product of a “dual-specifictetravalent full length binding protein”.

The present invention provides methods of expressing a dual variabledomain light chain and a dual variable domain heavy chain in a singlecell leading to a “primary product” of a “dual-specific tetravalent fulllength binding protein”, where the “primary product” is more than 50% ofall assembled protein, comprising a dual variable domain light chain anda dual variable domain heavy chain.

The present invention provides methods of expressing a dual variabledomain light chain and a dual variable domain heavy chain in a singlecell leading to a single “primary product” of a “dual-specifictetravalent full length binding protein”, where the “primary product” ismore than 75% of all assembled protein, comprising a dual variabledomain light chain and a dual variable domain heavy chain.

The present invention provides methods of expressing a dual variabledomain light chain and a dual variable domain heavy chain in a singlecell leading to a single “primary product” of a “dual-specifictetravalent full length binding protein”, where the “primary product” ismore than 90% of all assembled protein, comprising a dual variabledomain light chain and a dual variable domain heavy chain.

II. Derivatized DVD Binding Proteins

One embodiment provides a labeled binding protein wherein the bindingprotein of the invention is derivatized or linked to another functionalmolecule (e.g., another peptide or protein). For example, a labeledbinding protein of the invention can be derived by functionally linkinga binding protein of the invention (by chemical coupling, geneticfusion, noncovalent association or otherwise) to one or more othermolecular entities, such as another antibody (e.g., a bispecificantibody or a diabody), a detectable agent, a cytotoxic agent, apharmaceutical agent, and/or a protein or peptide that can mediateassociation of the binding protein with another molecule (such as astreptavidin core region or a polyhistidine tag).

Useful detectable agents with which a binding protein of the inventionmay be derivatized include fluorescent compounds. Exemplary fluorescentdetectable agents include fluorescein, fluorescein isothiocyanate,rhodamine, 5-dimethylamine-1-napthalenesulfonyl chloride, phycoerythrinand the like. A binding protein may also be derivatized with detectableenzymes, such as alkaline phosphatase, horseradish peroxidase, glucoseoxidase and the like. When a binding protein is derivatized with adetectable enzyme, it is detected by adding additional reagents that theenzyme uses to produce a detectable reaction product. For example, whenthe detectable agent horseradish peroxidase is present, the addition ofhydrogen peroxide and diaminobenzidine leads to a colored reactionproduct, which is detectable. A binding protein may also be derivatizedwith biotin, and detected through indirect measurement of avidin orstreptavidin binding.

Another embodiment of the invention provides a crystallized bindingprotein and formulations and compositions comprising such crystals. Inone embodiment the crystallized binding protein has a greater half-lifein vivo than the soluble counterpart of the binding protein. In anotherembodiment the binding protein retains biological activity aftercrystallization.

Crystallized binding protein of the invention may be produced accordingto methods known in the art and as disclosed in PCT Publication No. WO02072636.

Another embodiment of the invention provides a glycosylated bindingprotein wherein the antibody or antigen-binding portion thereofcomprises one or more carbohydrate residues. Nascent in vivo proteinproduction may undergo further processing, known as post-translationalmodification. In particular, sugar (glycosyl) residues may be addedenzymatically, a process known as glycosylation. The resulting proteinsbearing covalently linked oligosaccharide side chains are known asglycosylated proteins or glycoproteins. Antibodies are glycoproteinswith one or more carbohydrate residues in the Fc domain, as well as thevariable domain. Carbohydrate residues in the Fc domain have animportant effect on the effector function of the Fc domain, with minimaleffect on antigen binding or half-life of the antibody (Jefferis (2005)Biotechnol. Prog. 21:11-16). In contrast, glycosylation of the variabledomain may have an effect on the antigen binding activity of theantibody. Glycosylation in the variable domain may have a negativeeffect on antibody binding affinity, likely due to steric hindrance (Coet al. (1993) Mol. Immunol. 30:1361-1367), or result in increasedaffinity for the antigen (Wallick et al. (1988) Exp. Med. 168:1099-1109;Wright et al. (1991) EMBO J. 10:2717-2723).

One aspect of the present invention is directed to generatingglycosylation site mutants in which the O- or N-linked glycosylationsite of the binding protein has been mutated. One skilled in the art cangenerate such mutants using standard well-known technologies.Glycosylation site mutants that retain the biological activity but haveincreased or decreased binding activity are another object of thepresent invention.

In still another embodiment, the glycosylation of the antibody orantigen-binding portion of the invention is modified. For example, anaglycoslated antibody can be made (i.e., the antibody lacksglycosylation). Glycosylation can be altered to, for example, increasethe affinity of the antibody for antigen. Such carbohydratemodifications can be accomplished by, for example, altering one or moresites of glycosylation within the antibody sequence. For example, one ormore amino acid substitutions can be made that result in elimination ofone or more variable region glycosylation sites to thereby eliminateglycosylation at that site. Such aglycosylation may increase theaffinity of the antibody for antigen. Such an approach is described infurther detail in PCT Publication No. WO2003016466 and U.S. Pat. Nos.5,714,350 and 6,350,861.

Additionally or alternatively, a modified binding protein of theinvention can be made that has an altered type of glycosylation, such asa hypofucosylated antibody having reduced amounts of fucosyl residues(see Kanda et al. (2007) J. Biotechnol. 130(3):300-310) or an antibodyhaving increased bisecting GlcNAc structures. Such altered glycosylationpatterns have been demonstrated to increase the ADCC ability ofantibodies. Such carbohydrate modifications can be accomplished by, forexample, expressing the antibody in a host cell with alteredglycosylation machinery. Cells with altered glycosylation machinery havebeen described in the art and can be used as host cells in which toexpress recombinant antibodies of the invention to thereby produce anantibody with altered glycosylation. See, for example, Shields et al.(2002) J. Biol. Chem. 277:26733-26740; Umana et al. (1999) Nat. Biotech.17:176-1, as well as, European Patent No. EP 1,176,195; and PCTPublication Nos WO 03/035835 and WO 99/5434280.

Protein glycosylation depends on the amino acid sequence of the proteinof interest, as well as the host cell in which the protein is expressed.Different organisms may produce different glycosylation enzymes (e.g.,glycosyltransferases and glycosidases), and have different substrates(nucleotide sugars) available. Due to such factors, proteinglycosylation pattern, and composition of glycosyl residues, may differdepending on the host system in which the particular protein isexpressed. Glycosyl residues useful in the invention may include, butare not limited to, glucose, galactose, mannose, fucose,n-acetylglucosamine and sialic acid. In an embodiment, the glycosylatedbinding protein comprises glycosyl residues such that the glycosylationpattern is human.

It is known to those skilled in the art that differing proteinglycosylation may result in differing protein characteristics. Forinstance, the efficacy of a therapeutic protein produced in amicroorganism host, such as yeast, and glycosylated utilizing the yeastendogenous pathway may be reduced compared to that of the same proteinexpressed in a mammalian cell, such as a CHO cell line. Suchglycoproteins may also be immunogenic in humans and show reducedhalf-life in vivo after administration. Specific receptors in humans andother animals may recognize specific glycosyl residues and promote therapid clearance of the protein from the bloodstream. Other adverseeffects may include changes in protein folding, solubility,susceptibility to proteases, trafficking, transport,compartmentalization, secretion, recognition by other proteins orfactors, antigenicity, or allergenicity. Accordingly, a practitioner maychoose a therapeutic protein with a specific composition and pattern ofglycosylation, for example glycosylation composition and patternidentical, or at least similar, to that produced in human cells or inthe species-specific cells of the intended subject animal.

Expressing glycosylated proteins different from that of a host cell maybe achieved by genetically modifying the host cell to expressheterologous glycosylation enzymes. Using techniques known in the art apractitioner may generate antibodies or antigen-binding portions thereofexhibiting human protein glycosylation. For example, yeast strains havebeen genetically modified to express non-naturally occurringglycosylation enzymes such that glycosylated proteins (glycoproteins)produced in these yeast strains exhibit protein glycosylation identicalto that of animal cells, especially human cells (U.S. Pat. Nos.7,449,308 and 7,029,872 and PCT Publication No/WO2005/100584).

In addition to the binding proteins, the present invention is alsodirected to anti-idiotypic (anti-Id) antibodies specific for suchbinding proteins of the invention. An anti-Id antibody is an antibody,which recognizes unique determinants generally associated with theantigen-binding region of another antibody. The anti-Id can be preparedby immunizing an animal with the binding protein or a CDR containingregion thereof. The immunized animal will recognize, and respond to theidiotypic determinants of the immunizing antibody and produce an anti-Idantibody. It is readily apparent that it may be easier to generateanti-idiotypic antibodies to the two or more parent antibodiesincorporated into a DVD-Ig molecule; and confirm binding studies bymethods well recognized in the art (e.g., BIAcore, ELISA) to verify thatanti-idiotypic antibodies specific for the idiotype of each parentantibody also recognize the idiotype (e.g., antigen binding site) in thecontext of the DVD-Ig. The anti-idiotypic antibodies specific for eachof the two or more antigen binding sites of a DVD-Ig provide idealreagents to measure DVD-Ig concentrations of a human DVD-Ig in patientserum; DVD-Ig concentration assays can be established using a “sandwichassay ELISA format” with an antibody to a first antigen binding regionscoated on the solid phase (e.g., BIAcore chip, ELISA plate etc.), rinsedwith rinsing buffer, incubated with the serum sample, rinsed again andultimately incubated with another anti-idiotypic antibody to the anotherantigen binding site, itself labeled with an enzyme for quantitation ofthe binding reaction. In an embodiment, for a DVD-Ig with more than twodifferent binding sites, anti-idiotypic antibodies to the two outermostbinding sites (most distal and proximal from the constant region) willnot only help in determining the DVD-Ig concentration in human serum butalso document the integrity of the molecule in vivo. Each anti-Idantibody may also be used as an “immunogen” to induce an immune responsein yet another animal, producing a so-called anti-anti-Id antibody.

Further, it will be appreciated by one skilled in the art that a proteinof interest may be expressed using a library of host cells geneticallyengineered to express various glycosylation enzymes, such that memberhost cells of the library produce the protein of interest with variantglycosylation patterns. A practitioner may then select and isolate theprotein of interest with particular novel glycosylation patterns. In anembodiment, the protein having a particularly selected novelglycosylation pattern exhibits improved or altered biologicalproperties.

III. Uses of DVD-Ig

Given their ability to bind to two or more antigens the binding proteinsof the invention can be used to detect the antigens (e.g., in abiological sample, such as serum or plasma), using a conventionalimmunoassay, such as an enzyme linked immunosorbent assays (ELISA), aradioimmunoassay (RIA) or tissue immunohistochemistry. The DVD-Ig isdirectly or indirectly labeled with a detectable substance to facilitatedetection of the bound or unbound antibody. Suitable detectablesubstances include various enzymes, prosthetic groups, fluorescentmaterials, luminescent materials and radioactive materials. Examples ofsuitable enzymes include horseradish peroxidase, alkaline phosphatase,β-galactosidase, or acetylcholinesterase; examples of suitableprosthetic group complexes include streptavidin/biotin andavidin/biotin; examples of suitable fluorescent materials includeumbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine,dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin; anexample of a luminescent material includes luminol; and examples ofsuitable radioactive material include ³H, ¹⁴C, ³⁵S, ⁹⁰Y, ⁹⁹Tc, ¹¹¹In,¹²⁵I, ¹³¹I, ¹⁷⁷Lu, ¹⁶⁶Ho, or ¹⁵³Sm.

In an embodiment, the binding proteins of the invention neutralize theactivity of the antigens both in vitro and in vivo. Accordingly, suchDVD-Igs can be used to inhibit antigen activity, e.g., in a cell culturecontaining the antigens, in human subjects or in other mammaliansubjects having the antigens with which a binding protein of theinvention cross-reacts. In another embodiment, the invention provides amethod for reducing antigen activity in a subject suffering from adisease or disorder in which the antigen activity is detrimental. Abinding protein of the invention can be administered to a human subjectfor therapeutic purposes.

The term “a disorder in which antigen activity is detrimental” includesdiseases and other disorders in which the presence of the antigen in asubject suffering from the disorder has been shown to be or is suspectedof being either responsible for the pathophysiology of the disorder or afactor that contributes to a worsening of the disorder. Accordingly, adisorder in which antigen activity is detrimental is a disorder in whichreduction of antigen activity is expected to alleviate the symptomsand/or progression of the disorder. Such disorders may be evidenced, forexample, by an increase in the concentration of the antigen in abiological fluid of a subject suffering from the disorder (e.g., anincrease in the concentration of antigen in serum, plasma, synovialfluid, etc. of the subject). Non-limiting examples of disorders that canbe treated with the binding proteins of the invention include thosedisorders discussed below and in the section pertaining topharmaceutical compositions of the antibodies of the invention.

The DVD-Igs of the invention may bind one antigen or multiple antigens.Such antigens include, but are not limited to, the targets listed in thefollowing databases, which databases are incorporated herein byreference. These target databases include those listings:

Therapeutic targets (xin.cz3.nus.edu.sg/group/cjttd/ttd.asp);

Cytokines and cytokine receptors (www.cytokinewebfacts.com/,www.copewithcytokines.de/cope.cgi, and

cmbi.bjmu.edu.cn/cmbidata/cgf/CGF_Database/cytokine.medic.kumamoto-u.ac.jp/CFC/indexR.html);

Chemokines (cytokine.medic.kumamoto-u.ac.jp/CFC/CK/Chemokine.html);

Chemokine receptors and GPCRs(csp.medic.kumamoto-u.ac.jp/CSP/Receptor.html,http://www.gper.org/7tm/);

Olfactory Receptors (senselab.med.yale.edu/senselab/ORDB/default.asp);

Receptors (www.iuphar-db.org/iuphar-rd/list/index.htm);

Cancer targets (cged.hgc.jp/cgi-bin/input.cgi);

Secreted proteins as potential antibody targets (spd.cbi.pku.edu.cn/);

Protein kinases (spd.cbi.pku.edu.cn/), and

Human CD markers(content.labvelocity.com/tools/6/1226/CD_table_final_locked.pdf) and(Zola (2005) Blood 106:3123-6).

DVD-Igs are useful as therapeutic agents to simultaneously block twodifferent targets to enhance efficacy/safety and/or increase patientcoverage. Such targets may include soluble targets (e.g., TNF) and cellsurface receptor targets (e.g., VEGFR and EGFR). It can also be used toinduce redirected cytotoxicity between tumor cells and T cells (e.g.,Her2 and CD3) for cancer therapy, or between autoreactive cell andeffector cells for autoimmune disease or transplantation, or between anytarget cell and effector cell to eliminate disease-causing cells in anygiven disease.

In addition, DVD-Ig can be used to trigger receptor clustering andactivation when it is designed to target two different epitopes on thesame receptor. This may have benefit in making agonistic andantagonistic anti-GPCR therapeutics. In this case, DVD-Ig can be used totarget two different epitopes (including epitopes on both the loopregions and the extracellular domain) on one cell forclustering/signaling (two cell surface molecules) or signaling (on onemolecule). Similarly, a DVD-Ig molecule can be designed to triger CTLA-4ligation, and a negative signal by targeting two different epitopes (or2 copies of the same epitope) of CTLA-4 extracellular domain, leading todown regulation of the immune response. CTLA-4 is a clinically validatedtarget for therapeutic treatment of a number of immunological disorders.CTLA-4/B7 interactions negatively regulate T cell activation byattenuating cell cycle progression, IL-2 production, and proliferationof T cells following activation, and CTLA-4 (CD152) engagement candown-regulate T cell activation and promote the induction of immunetolerance. However, the strategy of attenuating T cell activation byagonistic antibody engagement of CTLA-4 has been unsuccessful sinceCTLA-4 activation requires ligation. The molecular interaction ofCTLA-4/B7 is in “skewed zipper” arrays, as demonstrated by crystalstructural analysis (Stamper (2001) Nature 410:608). However none of thecurrently available CTLA-4 binding reagents have ligation properties,including anti-CTLA-4 mAbs. There have been several attempts to addressthis issue. In one case, a cell member-bound single chain antibody wasgenerated, and significantly inhibited allogeneic rejection in mice(Hwang (2002) J. Immunol. 169:633). In a separate case, artificial APCsurface-linked single-chain antibody to CTLA-4 was generated anddemonstrated to attenuate T cell responses (Griffin (2000) J. Immunol.164:4433). In both cases, CTLA-4 ligation was achieved by closelylocalized member-bound antibodies in artificial systems. While theseexperiments provide proof-of-concept for immune down-regulation bytriggering CTLA-4 negative signaling, the reagents used in these reportsare not suitable for therapeutic use. To this end, CTLA-4 ligation maybe achieved by using a DVD-Ig molecule, which target two differentepitopes (or 2 copies of the same epitope) of CTLA-4 extracellulardomain. The rationale is that the distance spanning two binding sites ofan IgG, approximately 150-170 Å, is too large for active ligation ofCTLA-4 (30-50 Å between 2 CTLA-4 homodimer). However the distancebetween the two binding sites on DVD-Ig (one arm) is much shorter, alsoin the range of 30-50 Å, allowing proper ligation of CTLA-4.

Similarly, DVD-Ig can target two different members of a cell surfacereceptor complex (e.g., IL-12R alpha and beta). Furthermore, DVD-Ig cantarget CR1 and a soluble protein/pathogen to drive rapid clearance ofthe target soluble protein/pathogen.

Additionally, DVD-Igs of the invention can be employed fortissue-specific delivery (target a tissue marker and a disease mediatorfor enhanced local PK thus higher efficacy and/or lower toxicity),including intracellular delivery (targeting an internalizing receptorand an intracellular molecule), and delivering to the inside of thebrain (e.g., targeting transferrin receptor and a CNS disease mediatorfor crossing the blood-brain barrier). DVD-Ig can also serve as acarrier protein to deliver an antigen to a specific location via bindingto a non-neutralizing epitope of that antigen and also to increase thehalf-life of the antigen. Furthermore, DVD-Ig can be designed to eitherbe physically linked to medical devices implanted into patients ortarget these medical devices (see Burke et al. (2006) Adv. Drug Deliv.Rev. 58(3):37-446; Surface coatings for biological activation andfunctionalization of medical devices (see Hildebrand et al. (2006)Surface Coatings Technol. 200(22-23):6318-6324; Drug/device combinationsfor local drug therapies and infection prophylaxis (see Wu et al. (2006)Biomaterials 27(11):2450-2467); Mediation of the cytokine network in theimplantation of orthopedic devices (see Marques et al. BiodegradableSystems in Tissue Engineering and Regenerative Medicine (2005), pp.377-397). Briefly, directing appropriate types of cell to the site ofmedical implant may promote healing and restoring normal tissuefunction. Alternatively, inhibition of mediators (including but notlimited to cytokines), released upon device implantation by a DVDcoupled to or target to a device is also provided. For example, stentshave been used for years in interventional cardiology to clear blockedarteries and to improve the flow of blood to the heart muscle. However,traditional bare metal stents have been known to cause restenosis(re-narrowing of the artery in a treated area) in some patients and canlead to blood clots. Recently, an anti-CD34 antibody coated stent hasbeen described which reduced restenosis and prevents blood clots fromoccurring by capturing endothelial progenitor cells (EPC) circulatingthroughout the blood. Endothelial cells are cells that line bloodvessels, allowing blood to flow smoothly. The EPCs adhere to the hardsurface of the stent forming a smooth layer that not only promoteshealing but prevents restenosis and blood clots, complicationspreviously associated with the use of stents (Aoji et al. (2005) J. Am.Coll. Cardiol. 45(10):1574-9). In addition to improving outcomes forpatients requiring stents, there are also implications for patientsrequiring cardiovascular bypass surgery. For example, a prostheticvascular conduit (artificial artery) coated with anti-EPC antibodieswould eliminate the need to use arteries from patients legs or arms forbypass surgery grafts. This would reduce surgery and anesthesia times,which in turn will reduce coronary surgery deaths. DVD-Ig are designedin such a way that it binds to a cell surface marker (such as CD34) aswell as a protein (or an epitope of any kind, including but not limitedto proteins, lipids and polysaccharides) that has been coated on theimplanted device to facilitate the cell recruitment. Such approaches canalso be applied to other medical implants in general. Alternatively,DVD-Igs can be coated on medical devices and upon implantation andreleasing all DVDs from the device (or any other need which may requireadditional fresh DVD-Ig, including aging and denaturation of the alreadyloaded DVD-Ig) the device could be reloaded by systemic administrationof fresh DVD-Ig to the patient, where the DVD-Ig is designed to binds toa target of interest (a cytokine, a cell surface marker (such as CD34)etc.) with one set of binding sites and to a target coated on the device(including a protein, an epitope of any kind, including but not limitedto lipids, polysaccharides and polymers) with the other. This technologyhas the advantage of extending the usefulness of coated implants.

A. Use of DVD-Igs in Various Diseases

DVD-Ig molecules of the invention are also useful as therapeuticmolecules to treat various diseases. Such DVD molecules may bind one ormore targets involved in a specific disease. Examples of such targets invarious diseases are described below.

1. Human Autoimmune and Inflammatory Response

Many proteins have been implicated in general autoimmune andinflammatory responses, including C5, CCL1 (I-309), CCL11 (eotaxin),CCL13 (mcp-4), CCL15 (MIP-1d), CCL16 (HCC-4), CCL17 (TARC), CCL18(PARC), CCL19, CCL2 (mcp-1), CCL20 (MIP-3a), CCL21 (MIP-2), CCL23(MPIF-1), CCL24 (MPIF-2/eotaxin-2), CCL25 (TECK), CCL26, CCL3 (MIP-1a),CCL4 (MIP-1b), CCL5 (RANTES), CCL7 (mcp-3), CCL8 (mcp-2), CXCL1, CXCL10(IP-10), CXCL11 (1-TAC/IP-9), CXCL12 (SDF1), CXCL13, CXCL14, CXCL2,CXCL3, CXCL5 (ENA-78/LIX), CXCL6 (GCP-2), CXCL9, IL13, IL8, CCL13(mcp-4), CCR1, CCR2, CCR3, CCR4, CCR5, CCR6, CCR7, CCR8, CCR9, CX3CR1,IL8RA, XCR1 (CCXCR1), IFNA2, IL10, IL13, IL17C, IL1A, IL1B, IL1F10,IL1F5, IL1F6, IL1F7, IL1F8, IL1F9, IL22, IL5, IL8, IL9, LTA, LTB, MIF,SCYE1 (endothelial Monocyte-activating cytokine), SPP1, TNF, TNFSF5,IFNA2, IL10RA, IL10RB, IL13, IL13RA1, IL5RA, IL9, IL9R, ABCF1, BCL6, C3,C4A, CEBPB, CRP, ICEBERG, IL1R1, IL1RN, IL8RB, LTB4R, TOLLIP, FADD,IRAK1, IRAK2, MYD88, NCK2, TNFAIP3, TRADD, TRAF1, TRAF2, TRAF3, TRAF4,TRAF5, TRAF6, ACVR1, ACVR1B, ACVR2, ACVR2B, ACVRL1, CD28, CD3E, CD3G,CD3Z, CD69, CD80, CD86, CNR1, CTLA4, CYSLTR1, FCER1A, FCER2, FCGR3A,GPR44, HAVCR2, OPRD1, P2RX7, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8,TLR9, TLR10, BLR1, CCL1, CCL2, CCL3, CCL4, CCL5, CCL7, CCL8, CCL11,CCL13, CCL15, CCL16, CCL17, CCL18, CCL19, CCL20, CCL21, CCL22, CCL23,CCL24, CCL25, CCR1, CCR2, CCR3, CCR4, CCR5, CCR6, CCR7, CCR8, CCR9,CX3CL1, CX3CR1, CXCL1, CXCL2, CXCL3, CXCL5, CXCL6, CXCL10, CXCL11,CXCL12, CXCL13, CXCR4, GPR2, SCYE1, SDF2, XCL1, XCL2, XCR1, AMH, AMHR2,BMPR1A, BMPR1B, BMPR2, C19orf10 (IL27w), CER1, CSF1, CSF2, CSF3,DKFZp451J0118, FGF2, GFI1, IFNA 1, IFNB1, IFNG, IGF1, IL1A, IL1B, IL1R1,IL1R2, IL2, IL2RA, IL2RB, IL2RG, IL3, IL4, IL4R, IL5, IL5RA, IL6, IL6R,IL6ST, IL7, IL8, IL8RA, IL8RB, IL9, IL9R, IL10, IL10RA, IL10RB, IL11,IL11RA, IL12A, IL12B, IL12RB1, IL12RB2, IL13, IL13RA1, IL13RA2, IL15,IL15RA, IL16, IL17, IL17R, IL18, IL18R1, IL19, IL20, KITLG, LEP, LTA,LTB, LTB4R, LTB4R2, LTBR, MIF, NPPB, PDGFB, TBX21, TDGF1, TGFA, TGFB1,TGFB111, TGFB2, TGFB3, TGFB1, TGFBR1, TGFBR2, TGFBR3, TH1L, TNF,TNFRSF1A, TNFRSF1B, TNFRSF7, TNFRSF8, TNFRSF9, TNFRSF11A, TNFRSF21,TNFSF4, TNFSF5, TNFSF6, TNFSF11, VEGF, ZFPM2, and RNF110 (ZNF144). Inone aspect, DVD-Igs that bind one or more of the targets listed hereinare provided.

DVD Igs that bind the following pairs of targets to treat inflammatorydisease are contemplated: IL-1alpha (seq. 3) and IL-1beta (seq. 1);IL-1alpha (seq. 2) and IL-1beta (seq. 1); IL-1alpha (seq. 1) andIL-1beta (seq. 1); IL-1alpha (seq. 3) and IL-1beta (seq. 2); IL-1alpha(seq. 4) and IL-1beta (seq. 2); IL-1alpha (seq. 4) and IL-1beta (seq.3); IL-1alpha (seq. 4) and IL-1beta (seq. 4); IL-1alpha (seq. 4) andIL-1beta (seq. 5) (see Examples 2.1 to 2.8).

2. Asthma

Allergic asthma is characterized by the presence of eosinophilia, gobletcell metaplasia, epithelial cell alterations, airway hyperreactivity(AHR), and Th2 and Th1 cytokine expression, as well as elevated serumIgE levels. It is now widely accepted that airway inflammation is thekey factor underlying the pathogenesis of asthma, involving a complexinterplay of inflammatory cells such as T cells, B cells, eosinophils,mast cells and macrophages, and of their secreted mediators includingcytokines and chemokines. Corticosteroids are the most importantanti-inflammatory treatment for asthma today, however their mechanism ofaction is non-specific and safety concerns exist, especially in thejuvenile patient population. The development of more specific andtargeted therapies is therefore warranted. There is increasing evidencethat IL-13 in mice mimics many of the features of asthma, including AHR,mucus hypersecretion and airway fibrosis, independently of eosinophilicinflammation (Finotto et al. (2005) Int. Immunol. 17(8):993-1007;Padilla et al. (2005) J. Immunol. 174(12):8097-8105).

IL-13 has been implicated as having a pivotal role in causingpathological responses associated with asthma. The development ofanti-IL-13 mAb therapy to reduce the effects of IL-13 in the lung is anexciting new approach that offers considerable promise as a noveltreatment for asthma. However other mediators of differentialimmunological pathways are also involved in asthma pathogenesis, andblocking these mediators, in addition to IL-13, may offer additionaltherapeutic benefit. Such target pairs include, but are not limited to,IL-13 and a pro-inflammatory cytokine, such as tumor necrosis factor-α(TNF-α). TNF-α may amplify the inflammatory response in asthma and maybe linked to disease severity (McDonnell et al. (2001) Progr. Respir.Res. 31 (New Drugs for Asthma, Allergy and COPD):247-250). This suggeststhat blocking both IL-13 and TNF-α may have beneficial effects,particularly in severe airway disease. In another embodiment the DVD-Igof the invention binds the targets IL-13 and TNFα and is used fortreating asthma.

Animal models such as OVA-induced asthma mouse model, where bothinflammation and AHR can be assessed, are known in the art and may beused to determine the ability of various DVD-Ig molecules to treatasthma Animal models for studying asthma are disclosed in Coffman et al.(2005) J. Exp. Med. 201(12):1875-1879; Lloyd et al. (2001) Adv. Immunol.77:263-295; Boyce et al. (2005) J. Exp. Med. 201(12):1869-1873; andSnibson et al. (2005) J. Brit. Soc. Allergy Clin. Immunol. 35(2):146-52.In addition to routine safety assessments of these target pairs specifictests for the degree of immunosuppression may be warranted and helpfulin selecting the best target pairs (see Luster et al. (2004) Toxicol.92(1-3):229-43; Descotes et al. (1992) Dev. Biol. Standardiz. 77:99-102;Hart et al. (2001) J. Allergy and Clin. Immunol. 108(2):250-257).

Based on the rationale disclosed herein and using the same evaluationmodel for efficacy and safety other pairs of targets that DVD-Igmolecules can bind and be useful to treat asthma may be determined. Inan embodiment, such targets include, but are not limited to, IL-13 andIL-1beta, since IL-1beta is also implicated in inflammatory response inasthma; IL-13 and cytokines and chemokines that are involved ininflammation, such as IL-13 and IL-9; IL-13 and IL-4; IL-13 and IL-5;IL-13 and IL-25; IL-13 and TARC; IL-13 and MDC; IL-13 and MIF; IL-13 andTGF-β; IL-13 and LHR agonist; IL-13 and CL25; IL-13 and SPRR2a; IL-13and SPRR2b; and IL-13 and ADAMS. The present invention also providesDVD-Igs that bind one or more targets involved in asthma selected fromthe group consisting of CSF1 (MCSF), CSF2 (GM-CSF), CSF3 (GCSF), FGF2,IFNA1, IFNB1, IFNG, histamine and histamine receptors, IL1A, IL1B, IL2,IL3, IL4, IL5, IL6, IL7, IL8, IL9, IL10, IL11, IL12A, IL12B, IL13, IL14,IL15, IL16, IL17, IL18, IL19, KITLG, PDGFB, IL2RA, IL4R, IL5RA, IL8RA,IL8RB, IL12RB1, IL12RB2, IL13RA1, IL13RA2, IL18R1, TSLP, CCL1, CCL2,CCL3, CCL4, CCL5, CCL7, CCL8, CCL13, CCL17, CCL18, CCL19, CCL20, CCL22,CCL24, CX3CL1, CXCL1, CXCL2, CXCL3, XCL1, CCR2, CCR3, CCR4, CCR5, CCR6,CCR7, CCR8, CX3CR1, GPR2, XCR1, FOS, GATA3, JAK1, JAK3, STATE, TBX21,TGFB1, TNF, TNFSF6, YY1, CYSLTR1, FCER1A, FCER2, LTB4R, TB4R2, LTBR, andChitinase.

3. Rheumatoid Arthritis

Rheumatoid arthritis (RA), a systemic disease, is characterized by achronic inflammatory reaction in the synovium of joints and isassociated with degeneration of cartilage and erosion of juxta-articularbone. Many pro-inflammatory cytokines including TNF, chemokines, andgrowth factors are expressed in diseased joints. Systemic administrationof anti-TNF antibody or sTNFR fusion protein to mouse models of RA wasshown to be anti-inflammatory and joint protective. Clinicalinvestigations in which the activcity of TNF in RA patients was blockedwith intravenously administered infliximab (Harriman et al. (1999) Ann.Rheum. Dis. 58 Suppl 1:161-4), a chimeric anti-TNF mAb, has providedevidence that TNF regulates IL-6, IL-8, MCP-1, and VEGF production,recruitment of immune and inflammatory cells into joints, angiogenesis,and reduction of blood levels of matrix metalloproteinases-1 and -3. Abetter understanding of the inflammatory pathway in rheumatoid arthritishas led to identification of other therapeutic targets involved inrheumatoid arthritis. Promising treatments such as interleukin-6antagonists (IL-6 receptor antibody MRA, developed by Chugai, Roche (seeNishimoto et al. (2004) Arthritis Rheum. 50(6):1761-1769), CTLA4Ig(abatacept, Genovese et al. (2005) N. Engl. J. Med. 353:1114-23), andanti-B cell therapy (rituximab, Okamoto (2004) N. Engl. J. Med.351:1909) have already been tested in randomized controlled trials overthe past year. Other cytokines have been identified and have been shownto be of benefit in animal models, including interleukin-15 (therapeuticantibody HuMax-IL_(—)15, AMG 714 see Baslund et al. (2005) Arthrit.Rheum. 52(9):2686-2692), interleukin-17, and interleukin-18, andclinical trials of these agents are currently under way. Dual-specificantibody therapy, combining anti-TNF and another mediator, has greatpotential in enhancing clinical efficacy and/or patient coverage. Forexample, blocking both TNF and VEGF can potentially eradicateinflammation and angiogenesis, both of which are involved inpathophysiology of RA. Blocking other pairs of targets involved in RAincluding, but not limited to, TNF and IL-18; TNF and IL-12; TNF andIL-23; TNF and IL-1beta; TNF and MIF; TNF and IL-17; and TNF and IL-15with specific DVD Igs is also contemplated. In addition to routinesafety assessments of these target pairs, specific tests for the degreeof immunosuppression may be warranted and helpful in selecting the besttarget pairs (see Luster et al. (2004) Toxicol. 92(1-3):229-43; Descoteset al. (1992) Dev. Biol. Standard. 77:99-102; Hart et al. (2001) J.Allergy Clin. Immunol. 108(2):250-257). Whether a DVD Ig molecule willbe useful for the treatment of rheumatoid arthritis can be assessedusing pre-clinical animal RA models such as the collagen-inducedarthritis mouse model. Other useful models are also well known in theart (see Brand (2005) Comp. Med. 55(2):114-22). Based on thecross-reactivity of the parental antibodies for human and mouseothologues (e.g., reactivity for human and mouse TNF, human and mouseIL-15, etc.) validation studies in the mouse CIA model may be conductedwith “matched surrogate antibody” derived DVD-Ig molecules; briefly, aDVD-Ig based on two (or more) mouse target specific antibodies may bematched to the extent possible to the characteristics of the parentalhuman or humanized antibodies used for human DVD-Ig construction(similar affinity, similar neutralization potency, similar half-life,etc.).

4. SLE

The immunopathogenic hallmark of SLE is the polyclonal B cellactivation, which leads to hyperglobulinemia, autoantibody productionand immune complex formation. The fundamental abnormality appears to bethe failure of T cells to suppress the forbidden B cell clones due togeneralized T cell dysregulation. In addition, B and T-cell interactionis facilitated by several cytokines such as IL-10 as well asco-stimulatory molecules such as CD40 and CD40L, B7 and CD28 and CTLA-4,which initiate the second signal. These interactions together withimpaired phagocytic clearance of immune complexes and apoptoticmaterial, perpetuate the immune response with resultant tissue injury.The following targets may be involved in SLE and can potentially be usedfor a DVD-Ig approach for therapeutic intervention: B cell targetedtherapies: CD-20, CD-22, CD-19, CD28, CD4, CD80, HLA-DRA, IL10, IL2,IL4, TNFRSF5, TNFRSF6, TNFSF5, TNFSF6, BLR1, HDAC4, HDAC5, HDAC7A,HDAC9, ICOSL, IGBP1, MS4A1, RGS1, SLA2, CD81, IFNB1, IL10, TNFRSF5,TNFRSF7, TNFSF5, AICDA, BLNK, GALNAC4S-6ST, HDAC4, HDAC5, HDAC7A, HDAC9,IL10, IL11, IL4, INHA, INHBA, KLF6, TNFRSF7, CD28, CD38, CD69, CD80,CD83, CD86, DPP4, FCER2, IL2RA, TNFRSF8, TNFSF7, CD24, CD37, CD40, CD72,CD74, CD79A, CD79B, CR2, IL1R2, ITGA2, ITGA3, MS4A1, ST6GAL1, CD1C,CHST10, HLA-A, HLA-DRA, and NT5E.; co-stimulatory signals: CTLA4 orB7.1/B7.2; inhibition of B cell survival: BlyS, BAFF; Complementinactivation: C5; Cytokine modulation: the key principle is that the netbiologic response in any tissue is the result of a balance between locallevels of proinflammatory or anti-inflammatory cytokines (see Sfikakiset al. (2005) Curr. Opin. Rheumatol. 17:550-7). SLE is considered to bea Th-2 driven disease with documented elevations in serum IL-4, IL-6,IL-10. DVD Igs that bind one or more targets selected from the groupconsisting of IL-4, IL-6, IL-10, IFN-α, and TNF-α are also contemplated.Combination of targets discussed herein will enhance therapeuticefficacy for SLE which can be tested in a number of lupus preclinicalmodels (see Peng (2004) Methods Mol. Med. 102:227-72). Based on thecross-reactivity of the parental antibodies for human and mouseothologues (e.g., reactivity for human and mouse CD20, human and mouseInterferon alpha, etc.) validation studies in a mouse lupus model may beconducted with “matched surrogate antibody” derived DVD-Ig molecules;briefly, a DVD-Ig based two (or more) mouse target specific antibodiesmay be matched to the extent possible to the characteristics of theparental human or humanized antibodies used for human DVD-Igconstruction (similar affinity, similar neutralization potency, similarhalf-life, etc.).

5. Multiple Sclerosis

Multiple sclerosis (MS) is a complex human autoimmune-type disease witha predominantly unknown etiology. Immunologic destruction of myelinbasic protein (MBP) throughout the nervous system is the major pathologyof multiple sclerosis. MS is a disease of complex pathologies, whichinvolves infiltration by CD4+ and CD8+ T cells and of response withinthe central nervous system. Expression in the CNS of cytokines, reactivenitrogen species and costimulator molecules have all been described inMS. Of major consideration are immunological mechanisms that contributeto the development of autoimmunity. In particular, antigen expression,cytokine and leukocyte interactions, and regulatory T-cells, which helpbalance/modulate other T-cells such as Th1 and Th2 cells, are importantareas for therapeutic target identification.

IL-12 is a proinflammatory cytokine that is produced by APC and promotesdifferentiation of Th1 effector cells. IL-12 is produced in thedeveloping lesions of patients with MS as well as in EAE-affectedanimals. Previously it was shown that interference in IL-12 pathwayseffectively prevents EAE in rodents, and that in vivo neutralization ofIL-12p40 using an anti-IL-12 mAb has beneficial effects in themyelin-induced EAE model in common marmosets.

TWEAK is a member of the TNF family, constitutively expressed in thecentral nervous system (CNS), with pro-inflammatory, proliferative orapoptotic effects depending upon cell types. Its receptor, Fn14, isexpressed in CNS by endothelial cells, reactive astrocytes and neurons.TWEAK and Fn14 mRNA expression increased in spinal cord duringexperimental autoimmune encephalomyelitis (EAE). Anti-TWEAK antibodytreatment in myelin oligodendrocyte glycoprotein (MOG) induced EAE inC57BL/6 mice resulted in a reduction of disease severity and leukocyteinfiltration when mice were treated after the priming phase.

One aspect of the invention pertains to DVD-Ig molecules that bind oneor more, for example two, targets selected from the group consisting ofIL-12, TWEAK, IL-23, CXCL13, CD40, CD40L, IL-18, VEGF, VLA-4, TNF,CD45RB, CD200, IFNgamma, GM-CSF, FGF, C5, CD52, and CCR2. An embodimentincludes a dual-specific anti-IL-12/TWEAK DVD Ig as a therapeutic agentbeneficial for the treatment of MS.

Several animal models for assessing the usefulness of the DVD moleculesto treat MS are known in the art (see Steinman et al. (2005) TrendsImmunol. 26(11):565-71; Lublin et al. (1985) Springer SeminImmunopathol. 8(3):197-208; Genain et al. (1997) J. Mol. Med.75(3):187-97; Tuohy et al. (1999) J. Exp. Med. 189(7):1033-42; Owens etal. (1995) Neurol. Clin. 13(1):51-73; and Hart et al. (2005) J. Immunol.175(7):4761-8. Based on the cross-reactivity of the parental antibodiesfor human and animal species othologues (e.g., reactivity for human andmouse IL-12, human and mouse TWEAK etc.) validation studies in the mouseEAE model may be conducted with “matched surrogate antibody” derivedDVD-Ig molecules; briefly, a DVD-Ig based on two (or more) mouse targetspecific antibodies may be matched to the extent possible to thecharacteristics of the parental human or humanized antibodies used forhuman DVD-Ig construction (similar affinity, similar neutralizationpotency, similar half-life etc.). The same concept applies to animalmodels in other non-rodent species, where a “matched surrogate antibody”derived DVD-Ig would be selected for the anticipated pharmacology andpossibly safety studies. In addition to routine safety assessments ofthese target pairs specific tests for the degree of immunosuppressionmay be warranted and helpful in selecting the best target pairs (seeLuster et al. (1994) Toxicol. 92(1-3):229-43; Descotes et al. (1992)Devel. Biol. Standardiz. 77:99-102; Jones (2000) IDrugs 3(4):442-6).

6. Sepsis

The pathophysiology of sepsis is initiated by the outer membranecomponents of both gram-negative organisms (lipopolysaccharide [LPS],lipid A, endotoxin) and gram-positive organisms (lipoteichoic acid,peptidoglycan). These outer membrane components are able to bind to theCD14 receptor on the surface of monocytes. By virtue of the recentlydescribed toll-like receptors, a signal is then transmitted to the cell,leading to the eventual production of the proinflammatory cytokinestumor necrosis factor-alpha (TNF-alpha) and interleukin-1 (IL-1).Overwhelming inflammatory and immune responses are essential features ofseptic shock and play a central part in the pathogenesis of tissuedamage, multiple organ failure, and death induced by sepsis. Cytokines,especially tumor necrosis factor (TNF) and interleukin (IL-1), have beenshown to be critical mediators of septic shock. These cytokines have adirect toxic effect on tissues; they also activate phospholipase A2.These and other effects lead to increased concentrations ofplatelet-activating factor, promotion of nitric oxide synthase activity,promotion of tissue infiltration by neutrophils, and promotion ofneutrophil activity.

The treatment of sepsis and septic shock remains a clinical conundrum,and recent prospective trials with biological response modifiers (i.e.,anti-TNF and anti-MIF) aimed at the inflammatory response have shownonly modest clinical benefit. Recently, interest has shifted towardtherapies aimed at reversing the accompanying periods of immunesuppression. Studies in experimental animals and critically ill patientshave demonstrated that increased apoptosis of lymphoid organs and someparenchymal tissues contribute to this immune suppression, anergy, andorgan system dysfunction. During sepsis syndromes, lymphocyte apoptosiscan be triggered by the absence of IL-2 or by the release ofglucocorticoids, granzymes, or the so-called ‘death’ cytokines: tumornecrosis factor alpha or Fas ligand. Apoptosis proceeds viaauto-activation of cytosolic and/or mitochondrial caspases, which can beinfluenced by the pro- and anti-apoptotic members of the Bcl-2 family.In experimental animals, not only can treatment with inhibitors ofapoptosis prevent lymphoid cell apoptosis; it may also improve outcome.Although clinical trials with anti-apoptotic agents remain distant duein large part to technical difficulties associated with theiradministration and tissue targeting, inhibition of lymphocyte apoptosisrepresents an attractive therapeutic target for the septic patient.Likewise, a dual-specific agent targeting both inflammatory mediator andan apoptotic mediator, may have added benefit. One aspect of theinvention pertains to DVD-Igs that bind one or more targets involved insepsis, in an embodiment two targets, selected from the group consistingTNF, IL-1, MIF, IL-6, IL-8, IL-18, IL-12, IL-23, FasL, LPS, Toll-likereceptors, TLR-4, tissue factor, MIP-2, ADORA2A, CASP1, CASP4, IL-10,IL-1B, NFKB1, PROC, TNFRSF1A, CSF3, CCR3, IL1RN, MIF, NFKB1, PTAFR,TLR2, TLR4, GPR44, HMOX1, midkine, IRAK1, NFKB2, SERPINA1, SERPINE1, andTREM1. The efficacy of such DVD Igs for sepsis can be assessed inpreclinical animal models known in the art (see Buras et al. (2005) Nat.Rev. Drug Discov. 4(10):854-65 and Calandra et al. (2000) Nat. Med.6(2): 164-70).

7. Neurological Disorders

7.1. Neurodegenerative Diseases

Neurodegenerative diseases are either chronic in which case they areusually age-dependent or acute (e.g., stroke, traumatic brain injury,spinal cord injury, etc.). They are characterized by progressive loss ofneuronal functions (neuronal cell death, demyelination), loss ofmobility and loss of memory. Emerging knowledge of the mechanismsunderlying chronic neurodegenerative diseases (e g., Alzheimer'sdisease) show a complex etiology and a variety of factors have beenrecognized to contribute to their development and progression e.g., age,glycemic status, amyloid production and multimerization, accumulation ofadvanced glycation-end products (AGE) which bind to their receptor RAGE(receptor for AGE), increased brain oxidative stress, decreased cerebralblood flow, neuroinflammation including release of inflammatorycytokines and chemokines, neuronal dysfunction and microglialactivation. Thus these chronic neurodegenerative diseases represent acomplex interaction between multiple cell types and mediators. Treatmentstrategies for such diseases are limited and mostly constitute eitherblocking inflammatory processes with non-specific anti-inflammatoryagents (e.g., corticosteroids, COX inhibitors) or agents to preventneuron loss and/or synaptic functions. These treatments fail to stopdisease progression. Recent studies suggest that more targeted therapiessuch as antibodies to soluble A-b peptide (including the A-b oligomericforms) can not only help stop disease progression but may help maintainmemory as well. These preliminary observations suggest that specifictherapies targeting more than one disease mediator (e.g., A-b and apro-inflammatory cytokine such as TNF) may provide even bettertherapeutic efficacy for chronic neurodegenerative diseases thanobserved with targeting a single disease mechanism (e.g., soluble A-balone). Several animal models for assessing the usefulness of the DVD-Igmolecules to treat MS are known in the art (see Steinman et al. (2005)Trends Immunol. 26(11):565-71; Lublin et al. (1985) Springer Semin.Immunopathol. 8(3):197-208; Genain et al. (1997) J. Mol. Med.75(3):187-97; Tuohy et al. (1999) J. Exp. Med. 189(7):1033-42; Owens etal. (1995) Neurol. Clin. 13(1):51-73; and Hart et al. (2005) J. Immunol.175(7):4761-8. Based on the cross-reactivity of the parental antibodiesfor human and animal species othologues (e.g., reactivity for human andmouse IL-12, human and mouse TWEAK, etc.), validation studies in themouse EAE model may be conducted with “matched surrogate antibody”derived DVD-Ig molecules. Briefly, a DVD-Ig based on two (or more) mousetarget specific antibodies may be matched to the extent possible to thecharacteristics of the parental human or humanized antibodies used forhuman DVD-Ig construction (e.g., similar affinity, similarneutralization potency, similar half-life, etc.). The same conceptapplies to animal models in other non-rodent species, where a “matchedsurrogate antibody” derived DVD-Ig would be selected for the anticipatedpharmacology and possibly safety studies. In addition to routine safetyassessments of these target pairs specific tests for the degree ofimmunosuppression may be warranted and helpful in selecting the besttarget pairs (see Luster et al. (1994) Toxicol. 92(1-3):229-43; Descoteset al. (1992) Devel. Biol. Stand. 77:99-102; Jones (2000) IDrugs3(4):442-6).

The DVD-Ig molecules of the invention can bind one or more targetsinvolved in Chronic neurodegenerative diseases such as Alzheimers. Suchtargets include, but are not limited to, any mediator, soluble or cellsurface, implicated in AD pathogenesis, e.g., AGE (S100 A, amphoterin),pro-inflammatory cytokines (e.g., IL-1), chemokines (e.g., MCP 1),molecules that inhibit nerve regeneration (e.g., Nogo, RGM A), moleculesthat enhance neurite growth (neurotrophins) and molecules that canmediate transport at the blood brain barrier (e.g., transferrinreceptor, insulin receptor or RAGE). The efficacy of DVD-Ig moleculescan be validated in pre-clinical animal models such as the transgenicmice that over-express amyloid precursor protein or RAGE and developAlzheimer's disease-like symptoms. In addition, DVD-Ig molecules can beconstructed and tested for efficacy in the animal models and the besttherapeutic DVD-Ig can be selected for testing in human patients. DVD-Igmolecules can also be employed for treatment of other neurodegenerativediseases such as Parkinson's disease. Alpha-Synuclein is involved inParkinson's pathology. A DVD-Ig capable of targeting alpha-synuclein andinflammatory mediators such as TNF, IL-1, MCP-1 can prove effectivetherapy for Parkinson's disease and are contemplated in the invention.

7.2 Neuronal Regeneration and Spinal Cord Injury

Despite an increase in knowledge of the pathologic mechanisms, spinalcord injury (SCI) is still a devastating condition and represents amedical indication characterized by a high medical need. Most spinalcord injuries are contusion or compression injuries and the primaryinjury is usually followed by secondary injury mechanisms (inflammatorymediators e.g., cytokines and chemokines) that worsen the initial injuryand result in significant enlargement of the lesion area, sometimes morethan 10-fold. These primary and secondary mechanisms in SCI are verysimilar to those in brain injury caused by other means e.g., stroke. Nosatisfying treatment exists and high dose bolus injection ofmethylprednisolone (MP) is the only used therapy within a narrow timewindow of 8 h post injury. This treatment, however, is only intended toprevent secondary injury without causing any significant functionalrecovery. It is heavily criticized for the lack of unequivocal efficacyand severe adverse effects, like immunosuppression with subsequentinfections and severe histopathological muscle alterations. No otherdrugs, biologics or small molecules, stimulating the endogenousregenerative potential are approved, but promising treatment principlesand drug candidates have shown efficacy in animal models of SCI inrecent years. To a large extent the lack of functional recovery in humanSCI is caused by factors inhibiting neurite growth, at lesion sites, inscar tissue, in myelin as well as on injury-associated cells. Suchfactors are the myelin-associated proteins NogoA, OMgp and MAG, RGM A,the scar-associated CSPG (Chondroitin Sulfate Proteoglycans) andinhibitory factors on reactive astrocytes (some semaphorins andephrins). However, at the lesion site not only growth inhibitorymolecules are found but also neurite growth stimulating factors likeneurotrophins, laminin, L1 and others. This ensemble of neurite growthinhibitory and growth promoting molecules may explain that blockingsingle factors, like NogoA or RGM A, resulted in significant functionalrecovery in rodent SCI models, because a reduction of the inhibitoryinfluences could shift the balance from growth inhibition to growthpromotion. However, recoveries observed with blocking a single neuriteoutgrowth inhibitory molecule were not complete. To achieve faster andmore pronounced recoveries either blocking two neurite outgrowthinhibitory molecules, e.g., Nogo and RGM A, or blocking an neuriteoutgrowth inhibitory molecule and enhancing functions of a neuriteoutgrowth enhancing molecule, e.g., Nogo and neurotrophins, or blockinga neurite outgrowth inhibitory molecule, e.g., Nogo and apro-inflammatory molecule e.g., TNF, may be desirable (see McGee et al.(2003) Trends Neurosci. 26:193; Domeniconi et al. (2005) J. Neurol. Sci.233:43; Makwanal et al. (2005) FEBS J. 272:2628; Dickson (2002) Science298:1959; Teng, et al. (2005) J. Neurosci. Res. 79:273; Karnezis et al.(2004) Nature Neurosci. 7:736; Xu et al. (2004) J. Neurochem. 91:1018).

In one aspect, DVD-Igs that bind target pairs such as NgR and RGM A;NogoA and RGM A; MAG and RGM A; OMGp and RGM A; RGM A and RGM B; CSPGsand RGM A; aggrecan, midkine, neurocan, versican, phosphacan, Te38 andTNF-α; Aβ globulomer-specific antibodies combined with antibodiespromoting dendrite & axon sprouting are provided. Dendrite pathology isa very early sign of AD and it is known that NOGO A restricts dendritegrowth. One can combine such type of ab with any of the SCI-candidate(myelin-proteins) Ab. Other DVD-Ig targets may include any combinationof NgR-p75, NgR-Troy, NgR-Nogo66 (Nogo), NgR-Lingo, Lingo-Troy,Lingo-p75, MAG or Omgp. Additionally, targets may also include anymediator, soluble or cell surface, implicated in inhibition of neurite,e.g., Nogo, Ompg, MAG, RGM A, semaphorins, ephrins, soluble A-b,pro-inflammatory cytokines (e.g., IL-1), chemokines (e.g., MIP 1a),molecules that inhibit nerve regeneration. The efficacy ofanti-nogo/anti-RGM A or similar DVD-Ig molecules can be validated inpre-clinical animal models of spinal cord injury. In addition, theseDVD-Ig molecules can be constructed and tested for efficacy in theanimal models and the best therapeutic DVD-Ig can be selected fortesting in human patients. In addition, DVD-Ig molecules can beconstructed that target two distinct ligand binding sites on a singlereceptor, e.g., Nogo receptor which binds three ligand Nogo, Ompg, andMAG and RAGE that binds A-b and S100 A. Furthermore, neurite outgrowthinhibitors, e.g., nogo and nogo receptor, also play a role in preventingnerve regeneration in immunological diseases like multiple sclerosisInhibition of nogo-nogo receptor interaction has been shown to enhancerecovery in animal models of multiple sclerosis. Therefore, DVD-Igmolecules that can block the function of one immune mediator, e.g., acytokine like IL-12 and a neurite outgrowth inhibitor molecule eg nogoor RGM may offer faster and greater efficacy than blocking either animmune or an neurite outgrowth inhibitor molecule alone.

In general, antibodies do not cross the blood brain barrier (BBB) in anefficient and relevant manner. However, in certain neurologic diseases,e.g., stroke, traumatic brain injury, multiple sclerosis, etc., the BBBmay be compromised and allows for increased penetration of DVD-Igs andantibodies into the brain. In other neurological conditions, where BBBleakage is not occurring, one may employ the targeting of endogenoustransport systems, including carrier-mediated transporters such asglucose and amino acid carriers and receptor-mediatedtranscytosis-mediating cell structures/receptors at the vascularendothelium of the BBB, thus enabling trans-BBB transport of the DVD-Ig.Structures at the BBB enabling such transport include but are notlimited to the insulin receptor, transferrin receptor, LRP and RAGE. Inaddition, strategies enable the use of DVD-Igs also as shuttles totransport potential drugs into the CNS including low molecular weightdrugs, nanoparticles and nucleic acids (Coloma et al. (2000) Pharm Res.17(3):266-74; Boado et al. (2007) Bioconjug. Chem. 18(2):447-55).

8. Oncological Disorders

Monoclonal antibody therapy has emerged as an important therapeuticmodality for cancer (von Mehren et al. (2003) Annu. Rev. Med.54:343-69). Antibodies may exert antitumor effects by inducingapoptosis, redirected cytotoxicity, interfering with ligand-receptorinteractions, or preventing the expression of proteins that are criticalto the neoplastic phenotype. In addition, antibodies can targetcomponents of the tumor microenvironment, perturbing vital structuressuch as the formation of tumor-associated vasculature. Antibodies canalso target receptors whose ligands are growth factors, such as theepidermal growth factor receptor. The antibody thus inhibits naturalligands that stimulate cell growth from binding to targeted tumor cells.Alternatively, antibodies may induce an anti-idiotype network,complement-mediated cytotoxicity, or antibody-dependent cellularcytotoxicity (ADCC). The use of dual-specific antibody that targets twoseparate tumor mediators will likely give additional benefit compared toa mono-specific therapy.

In another embodiment, a DVD-Ig of the invention binds VEGF andphosphatidylserine; VEGF and ErbB3; VEGF and PLGF; VEGF and ROBO4; VEGFand BSG2; VEGF and CDCP1; VEGF and ANPEP; VEGF and c-MET; HER-2 andERB3; HER-2 and BSG2; HER-2 and CDCP1; HER-2 and ANPEP; EGFR and CD64;EGFR and BSG2; EGFR and CDCP1; EGFR and ANPEP; IGF1R and PDGFR; IGF1Rand VEGF; IGF1R and CD20; CD20 and CD74; CD20 and CD30; CD20 and DR4;CD20 and VEGFR2; CD20 and CD52; CD20 and CD4; HGF and c-MET; HGF andNRP1; HGF and phosphatidylserine; ErbB3 and IGF1R; ErbB3 and IGF1,2;c-Met and Her-2; c-Met and NRP1; c-Met and IGF1R; IGF1,2 and PDGFR;IGF1,2 and CD20; IGF1,2 and IGF1R; IGF2 and EGFR; IGF2 and HER2; IGF2and CD20; IGF2 and VEGF; IGF2 and IGF1R; IGF1 and IGF2; PDGFRa andVEGFR2; PDGFRa and PLGF; PDGFRa and VEGF; PDGFRa and c-Met; PDGFRa andEGFR; PDGFRb and VEGFR2; PDGFRb and c-Met; PDGFRb and EGFR; RON andc-Met; RON and MTSP1; RON and MSP; RON and CDCP1; VGFR1 and PLGF; VGFR1and RON; VGFR1 and EGFR; VEGFR2 and PLGF; VEGFR2 and NRP1; VEGFR2 andRON; VEGFR2 and DLL4; VEGFR2 and EGFR; VEGFR2 and ROBO4; VEGFR2 andCD55; LPA and SIP; EPHB2 and RON; CTLA4 and VEGF; CD3 and EPCAM; CD40and IL6; CD40 and IGF; CD40 and CD56; CD40 and CD70; CD40 and VEGFR1;CD40 and DR5; CD40 and DR4; CD40 and APRIL; CD40 and BCMA; CD40 andRANKL; CD28 and MAPG; CD80 and CD40; CD80 and CD30; CD80 and CD33; CD80and CD74; CD80 and CD2; CD80 and CD3; CD80 and CD19; CD80 and CD4; CD80and CD52; CD80 and VEGF; CD80 and DR5; CD80 and VEGFR2; CD22 and CD20;CD22 and CD80; CD22 and CD40; CD22 and CD23; CD22 and CD33; CD22 andCD74; CD22 and CD19; CD22 and DR5; CD22 and DR4; CD22 and VEGF; CD22 andCD52; CD30 and CD20; CD30 and CD22; CD30 and CD23; CD30 and CD40; CD30and VEGF; CD30 and CD74; CD30 and CD19; CD30 and DR5; CD30 and DR4; CD30and VEGFR2; CD30 and CD52; CD30 and CD4; CD138 and RANKL; CD33 and FTL3;CD33 and VEGF; CD33 and VEGFR2; CD33 and CD44; CD33 and DR4; CD33 andDR5; DR4 and CD137; DR4 and IGF1,2; DR4 and IGF1R; DR4 and DR5; DR5 andCD40; DR5 and CD137; DR5 and CD20; DR5 and EGFR; DR5 and IGF1,2; DR5 andIGFR, DR5 and HER-2, and EGFR and DLL4. Other target combinationsinclude one or more members of the EGF/erb-2/erb-3 family. Other targets(one or more) involved in oncological diseases that DVD Igs may bindinclude, but are not limited to those selected from the group consistingof: CD52, CD20, CD19, CD3, CD4, CD8, BMP6, IL12A, IL1A, IL1B, IL2, IL24,INHA, TNF, TNFSF10, BMP6, EGF, FGF1, FGF10, FGF11, FGF12, FGF13, FGF14,FGF16, FGF17, FGF18, FGF19, FGF2, FGF20, FGF21, FGF22, FGF23, FGF3,FGF4, FGF5, FGF6, FGF7, FGF8, FGF9, GRP, IGF1, IGF2, IL12A, IL1A, IL1B,IL2, INHA, TGFA, TGFB1, TGFB2, TGFB3, VEGF, CDK2, FGF10, FGF18, FGF2,FGF4, FGF7, IGF1R, IL2, BCL2, CD164, CDKN1A, CDKN1B, CDKN1C, CDKN2A,CDKN2B, CDKN2C, CDKN3, GNRH1, IGFBP6, IL1A, IL1B, ODZ1, PAWR, PLG,TGFB1I1, AR, BRCA1, CDK3, CDK4, CDK5, CDK6, CDK7, CDK9, E2F1, EGFR,ENO1, ERBB2, ESR1, ESR2, IGFBP3, IGFBP6, IL2, INSL4, MYC, NOX5, NR6A1,PAP, PCNA, PRKCQ, PRKD1, PRL, TP53, FGF22, FGF23, FGF9, IGFBP3, IL2,INHA, KLK6, TP53, CHGB, GNRH1, IGF1, IGF2, INHA, INSL3, INSL4, PRL,KLK6, SHBG, NR1D1, NR1H3, NR1I3, NR2F6, NR4A3, ESR1, ESR2, NR0B1, NR0B2,NR1D2, NR1H2, NR1H4, NR1I2, NR2C1, NR2C2, NR2E1, NR2E3, NR2F1, NR2F2,NR3C1, NR3C2, NR4A1, NR4A2, NR5A1, NR5A2, NR6A1, PGR, RARB, FGF1, FGF2,FGF6, KLK3, KRT1, APOC1, BRCA1, CHGA, CHGB, CLU, COL1A1, COL6A1, EGF,ERBB2, ERK8, FGF1, FGF10, FGF11, FGF13, FGF14, FGF16, FGF17, FGF18,FGF2, FGF20, FGF21, FGF22, FGF23, FGF3, FGF4, FGF5, FGF6, FGF7, FGF8,FGF9, GNRH1, IGF1, IGF2, IGFBP3, IGFBP6, IL12A, IL1A, IL1B, IL2, IL24,INHA, INSL3, INSL4, KLK10, KLK12, KLK13, KLK14, KLK15, KLK3, KLK4, KLK5,KLK6, KLK9, MMP2, MMP9, MSMB, NTN4, ODZ1, PAP, PLAU, PRL, PSAP,SERPINA3, SHBG, TGFA, TIMP3, CD44, CDH1, CDH10, CDH19, CDH20, CDH7,CDH9, CDH1, CDH10, CDH13, CDH18, CDH19, CDH20, CDH7, CDH8, CDH9, ROBO2,CD44, ILK, ITGA1, APC, CD164, COL6A1, MTSS1, PAP, TGFB1I1, AGR2, AIG1,AKAP1, AKAP2, CANT1, CAV1, CDH12, CLDN3, CLN3, CYB5, CYC1, DAB2IP, DES,DNCL1, ELAC2, ENO2, ENO3, FASN, FLJ12584, FLJ25530, GAGEB1, GAGEC1,GGT1, GSTP1, HIP1, HUMCYT2A, IL29, K6HF, KAI1, KRT2A, MIB1, PART1, PATE,PCA3, PIAS2, PIK3CG, PPID, PR1, PSCA, SLC2A2, SLC33A1, SLC43A1, STEAP,STEAP2, TPM1, TPM2, TRPC6, ANGPT1, ANGPT2, ANPEP, ECGF1, EREG, FGF1,FGF2, FIGF, FLT1, JAG1, KDR, LAMAS, NRP1, NRP2, PGF, PLXDC1, STAB1,VEGF, VEGFC, ANGPTL3, BAI1, COL4A3, IL8, LAMAS, NRP1, NRP2, STAB1,ANGPTL4, PECAM1, PF4, PROK2, SERPINF1, TNFAIP2, CCL11, CCL2, CXCL1,CXCL10, CXCL3, CXCL5, CXCL6, CXCL9, IFNA1, IFNB1, IFNG, IL1B, IL6, MDK,EDG1, EFNA1, EFNA3, EFNB2, EGF, EPHB4, FGFR3, HGF, IGF1, ITGB3, PDGFA,TEK, TGFA, TGFB1, TGFB2, TGFBR1, CCL2, CDH5, COL18A1, EDG1, ENG, ITGAV,ITGB3, THBS1, THBS2, BAD, BAG1, BCL2, CCNA1, CCNA2, CCND1, CCNE1, CCNE2,CDH1 (E-cadherin), CDKN1B (p27Kipl), CDKN2A (p161NK4a), COL6A1, CTNNB1(b-catenin), CTSB (cathepsin B), ERBB2 (Her-2), ESR1, ESR2, F3 (TF),FOSL1 (FRA-1), GATA3, GSN (Gelsolin), IGFBP2, IL2RA, IL6, IL6R, IL6ST(glycoprotein 130), ITGA6 (a6 integrin), JUN, KLK5, KRT19, MAP2K7(c-Jun), MKI67 (Ki-67), NGFB (NGF), NGFR, NME1 (NM23A), PGR, PLAU (uPA),PTEN, SERPINB5 (maspin), SERPINE1 (PAI-1), TGFA, THBS1(thrombospondin-1), TIE (Tie-1), TNFRSF6 (Fas), TNFSF6 (FasL), TOP2A(topoisomerase Ea), TP53, AZGP1 (zinc-a-glycoprotein), BPAG1 (plectin),CDKN1A (p21Wap1/Cip1), CLDN7 (claudin-7), CLU (clusterin), ERBB2(Her-2), FGF1, FLRT1 (fibronectin), GABRP (GABAa), GNAS1, ID2, ITGA6 (a6integrin), ITGB4 (b 4 integrin), KLF5 (GC Box BP), KRT19 (Keratin 19),KRTHB6 (hair-specific type II keratin), MACMARCKS, MT3(metallothionectin-III), MUC1 (mucin), PTGS2 (COX-2), RAC2 (p21Rac2),S100A2, SCGB1D2 (lipophilin B), SCGB2A1 (mammaglobin 2), SCGB2A2(mammaglobin 1), SPRR1B (Spr1), THBS1, THBS2, THBS4, and TNFAIP2 (B94),RON, c-Met, CD64, DLL4, PLGF, CTLA4, phophatidylserine, ROBO4, CD80,CD22, CD40, CD23, CD28, CD80, CD55, CD38, CD70, CD74, CD30, CD138, CD56,CD33, CD2, CD137, DR4, DRS, RANKL, VEGFR2, PDGFR, VEGFR1, MTSP1, MSP,EPHB2, EPHA1, EPHA2, EpCAM, PGE2, NKG2D, LPA, SIP, APRIL, BCMA, MAPG,FLT3, PDGFR alpha, PDGFR beta, ROR1, PSMA, PSCA, SCD1, and CD59.

IV. Pharmaceutical Compositions

The invention also provides pharmaceutical compositions comprising abinding protein, of the invention and a pharmaceutically acceptablecarrier. The pharmaceutical compositions comprising binding proteins ofthe invention are for use in, but not limited to, diagnosing, detecting,or monitoring a disorder, in preventing (e g., inhibiting or delayingthe onset of a disease, disorder, or other condition), treating,managing, or ameliorating of a disorder or one or more symptoms thereof,and/or in research. In a specific embodiment, a composition comprisesone or more binding proteins of the invention. In another embodiment,the pharmaceutical composition comprises one or more binding proteins ofthe invention and one or more prophylactic or therapeutic agents otherthan binding proteins of the invention for treating a disorder. In anembodiment, the prophylactic or therapeutic agents are useful for orhave been or currently are being used in the prevention, treatment,management, or amelioration of a disorder or one or more symptomsthereof. In accordance with these embodiments, the composition mayfurther comprise a carrier, diluent or excipient.

The binding proteins of the invention can be incorporated intopharmaceutical compositions suitable for administration to a subject.Typically, the pharmaceutical composition comprises a binding protein ofthe invention and a pharmaceutically acceptable carrier. The term“pharmaceutically acceptable carrier” includes any and all solvents,dispersion media, coatings, antibacterial and antifungal agents,isotonic and absorption delaying agents, and the like that arephysiologically compatible. Examples of pharmaceutically acceptablecarriers include one or more of water, saline, phosphate bufferedsaline, dextrose, glycerol, ethanol and the like, as well ascombinations thereof. In some embodiments, isotonic agents, for example,sugars, polyalcohols such as mannitol, sorbitol, or sodium chloride, areincluded in the composition. Pharmaceutically acceptable carriers mayfurther comprise minor amounts of auxiliary substances such as wettingor emulsifying agents, preservatives or buffers, which enhance the shelflife or effectiveness of the antibody or antibody portion.

Various delivery systems are known and can be used to administer one ormore antibodies of the invention or the combination of one or moreantibodies of the invention and a prophylactic agent or therapeuticagent useful for preventing, managing, treating, or ameliorating adisorder or one or more symptoms thereof, e.g., encapsulation inliposomes, microparticles, microcapsules, recombinant cells capable ofexpressing the antibody or antibody fragment, receptor-mediatedendocytosis (see, e.g., Wu and Wu (1987) J. Biol. Chem. 262:4429-4432),construction of a nucleic acid as part of a retroviral or other vector,etc. Methods of administering a prophylactic or therapeutic agent of theinvention include, but are not limited to, parenteral administration(e.g., intradermal, intramuscular, intraperitoneal, intravenous andsubcutaneous), epidural administration, intratumoral administration, andmucosal administration (e.g., intranasal and oral routes). In addition,pulmonary administration can be employed, e.g., by use of an inhaler ornebulizer, and a formulation with an aerosolizing agent. See, e.g., U.S.Pat. Nos. 6,019,968; 5,985,320; 5,985,309; 5,934,272; 5,874,064;5,855,913; 5,290,540; and 4,880,078; and PCT Publication Nos. WO92/19244; WO 97/32572; WO 97/44013; WO 98/31346; and WO 99/66903. In oneembodiment, a binding protein of the invention, combination therapy, ora composition of the invention is administered using Alkermes AIR®pulmonary drug delivery technology (Alkermes, Inc., Cambridge, Mass.).In a specific embodiment, prophylactic or therapeutic agents of theinvention are administered intramuscularly, intravenously,intratumorally, orally, intranasally, pulmonary, or subcutaneously. Theprophylactic or therapeutic agents may be administered by any convenientroute, for example by infusion or bolus injection, by absorption throughepithelial or mucocutaneous linings (e.g., oral mucosa, rectal andintestinal mucosa, etc.) and may be administered together with otherbiologically active agents. Administration can be systemic or local.

In an embodiment, specific binding of antibody-coupled carbon nanotubes(CNTs) to tumor cells in vitro, followed by their highly specificablation with near-infrared (NIR) light can be used to target tumorcells. For example, biotinylated polar lipids can be used to preparestable, biocompatible, noncytotoxic CNT dispersions that are thenattached to one or two different neutralite avidin-derivatized DVD-Igsdirected against one or more tumor antigens (e.g., CD22) (Chakravarty etal. (2008) Proc. Natl. Acad. Sci. USA 105:8697-8702.

In a specific embodiment, it may be desirable to administer theprophylactic or therapeutic agents of the invention locally to the areain need of treatment; this may be achieved by, for example, and not byway of limitation, local infusion, by injection, or by means of animplant, said implant being of a porous or non-porous material,including membranes and matrices, such as sialastic membranes, polymers,fibrous matrices (e.g., Tissuel®), or collagen matrices. In oneembodiment, an effective amount of one or more antibodies of theinvention antagonists is administered locally to the affected area to asubject to prevent, treat, manage, and/or ameliorate a disorder or asymptom thereof. In another embodiment, an effective amount of one ormore antibodies of the invention is administered locally to the affectedarea in combination with an effective amount of one or more therapies(e.g., one or more prophylactic or therapeutic agents) other than abinding protein of the invention of a subject to prevent, treat, manage,and/or ameliorate a disorder or one or more symptoms thereof.

In another embodiment, the prophylactic or therapeutic agent can bedelivered in a controlled release or sustained release system. In oneembodiment, a pump may be used to achieve controlled or sustainedrelease (see Langer, supra; Sefton (1987) CRC Crit. Ref Biomed. Eng.14:20; Buchwald et al. (1980) Surgery 88:507; Saudek et al. (1989) N.Engl. J. Med. 321:574). In another embodiment, polymeric materials canbe used to achieve controlled or sustained release of the therapies ofthe invention (see, e.g., Medical Applications of Controlled Release,Langer and Wise (eds.), CRC Pres., Boca Raton, Fla. (1974); ControlledDrug Bioavailability, Drug Product Design and Performance, Smolen andBall (eds.), Wiley, New York (1984); Ranger and Peppas (1983) J.,Macromol. Sci. Rev. Macromol. Chem. 23:61; Levy et al. (1985) Science228:190; During et al. (1989) Ann. Neurol. 25:351; Howard et al. (1989)J. Neurosurg. 71:105); U.S. Pat. Nos. 5,679,377; 5,916,597; 5,912,015;5,989,463; 5,128,326; PCT Publication No. WO 99/15154 and WO 99/20253.Examples of polymers used in sustained release formulations include, butare not limited to, poly(2-hydroxy ethyl methacrylate), poly(methylmethacrylate), poly(acrylic acid), poly(ethylene-co-vinyl acetate),poly(methacrylic acid), polyglycolides (PLG), polyanhydrides,poly(N-vinyl pyrrolidone), poly(vinyl alcohol), polyacrylamide,poly(ethylene glycol), polylactides (PLA), poly(lactide-co-glycolides)(PLGA), and polyorthoesters. In an embodiment, the polymer used in asustained release formulation is inert, free of leachable impurities,stable on storage, sterile, and biodegradable. In yet anotherembodiment, a controlled or sustained release system can be placed inproximity of the prophylactic or therapeutic target, thus requiring onlya fraction of the systemic dose (see, e.g., Goodson (1984) in MedicalApplications of Controlled Release, supra, 2:115-138).

Controlled release systems are discussed in the review by Langer (1990)Science 249:1527-1533). Any technique known to one of skill in the artcan be used to produce sustained release formulations comprising one ormore therapeutic agents of the invention. See, e.g., U.S. Pat. No.4,526,938, PCT Publication Nos. WO 91/05548 and WO 96/20698, Ning et al.(1996) Radiother. Oncol. 39:179-189, Song et al. (1995) PDA J. Pharm.Sci. Technol. 50:372-397; Cleek et al. (1997) Pro. Int'l. Symp. Control.Rel. Bioact. Mater. 24:853-854; and Lam et al. (1997) Proc. Int'l. Symp.Control Rel. Bioact. Mater. 24:759-760.

In a specific embodiment, where the composition of the invention is anucleic acid encoding a prophylactic or therapeutic agent, the nucleicacid can be administered in vivo to promote expression of its encodedprophylactic or therapeutic agent, by constructing it as part of anappropriate nucleic acid expression vector and administering it so thatit becomes intracellular, e.g., by use of a retroviral vector (see U.S.Pat. No. 4,980,286), or by direct injection, or by use of microparticlebombardment (e.g., a gene gun; Biolistic, Dupont), or coating withlipids or cell-surface receptors or transfecting agents, or byadministering it in linkage to a homeobox-like peptide which is known toenter the nucleus (see, e.g., Joliot et al. (1991) Proc. Natl. Acad.Sci. USA 88:1864-1868). Alternatively, a nucleic acid can be introducedintracellularly and incorporated within host cell DNA for expression byhomologous recombination.

A pharmaceutical composition of the invention is formulated to becompatible with its intended route of administration. Examples of routesof administration include, but are not limited to, parenteral, e.g.,intravenous, intradermal, subcutaneous, oral, intranasal (e.g.,inhalation), transdermal (e.g., topical), transmucosal, and rectaladministration. In a specific embodiment, the composition is formulatedin accordance with routine procedures as a pharmaceutical compositionadapted for intravenous, subcutaneous, intramuscular, oral, intranasal,or topical administration to human beings. Typically, compositions forintravenous administration are solutions in sterile isotonic aqueousbuffer. Where necessary, the composition may also include a solubilizingagent and a local anesthetic such as lignocamne to ease pain at the siteof the injection.

If the compositions of the invention are to be administered topically,the compositions can be formulated in the form of an ointment, cream,transdermal patch, lotion, gel, shampoo, spray, aerosol, solution,emulsion, or other form well-known to one of skill in the art. See,e.g., Remington's Pharmaceutical Sciences and Introduction toPharmaceutical Dosage Forms, 19th ed., Mack Pub. Co., Easton, Pa.(1995). In an embodiment, for non-sprayable topical dosage forms,viscous to semi-solid or solid forms comprising a carrier or one or moreexcipients compatible with topical application and having a dynamicviscosity greater than water are employed. Suitable formulationsinclude, without limitation, solutions, suspensions, emulsions, creams,ointments, powders, liniments, salves, and the like, which are, ifdesired, sterilized or mixed with auxiliary agents (e.g., preservatives,stabilizers, wetting agents, buffers, or salts) for influencing variousproperties, such as, for example, osmotic pressure. Other suitabletopical dosage forms include sprayable aerosol preparations wherein theactive ingredient, in an embodiment, in combination with a solid orliquid inert carrier, is packaged in a mixture with a pressurizedvolatile (e.g., a gaseous propellant, such as freon) or in a squeezebottle. Moisturizers or humectants can also be added to pharmaceuticalcompositions and dosage forms if desired. Examples of such additionalingredients are well-known in the art.

If the method of the invention comprises intranasal administration of acomposition, the composition can be formulated in an aerosol form,spray, mist or in the form of drops. In particular, prophylactic ortherapeutic agents for use according to the present invention can beconveniently delivered in the form of an aerosol spray presentation frompressurized packs or a nebuliser, with the use of a suitable propellant(e.g., dichlorodifluoromethane, trichlorofluoromethane,dichlorotetrafluoroethane, carbon dioxide or other suitable gas). In thecase of a pressurized aerosol the dosage unit may be determined byproviding a valve to deliver a metered amount. Capsules and cartridges(composed of, e.g., gelatin) for use in an inhaler or insufflator may beformulated containing a powder mix of the compound and a suitable powderbase such as lactose or starch.

If the method of the invention comprises oral administration,compositions can be formulated orally in the form of tablets, capsules,cachets, gelcaps, solutions, suspensions, and the like. Tablets orcapsules can be prepared by conventional means with pharmaceuticallyacceptable excipients such as binding agents (e.g., pregelatinised maizestarch, polyvinylpyrrolidone, or hydroxypropyl methylcellulose); fillers(e.g., lactose, microcrystalline cellulose, or calcium hydrogenphosphate); lubricants (e.g., magnesium stearate, talc, or silica);disintegrants (e.g., potato starch or sodium starch glycolate); orwetting agents (e.g., sodium lauryl sulphate). The tablets may be coatedby methods well-known in the art. Liquid preparations for oraladministration may take the form of, but not limited to, solutions,syrups or suspensions, or they may be presented as a dry product forconstitution with water or other suitable vehicle before use. Suchliquid preparations may be prepared by conventional means withpharmaceutically acceptable additives such as suspending agents (e.g.,sorbitol syrup, cellulose derivatives, or hydrogenated edible fats);emulsifying agents (e.g., lecithin or acacia); non-aqueous vehicles(e.g., almond oil, oily esters, ethyl alcohol, or fractionated vegetableoils); and preservatives (e.g., methyl or propyl-p-hydroxybenzoates orsorbic acid). The preparations may also contain buffer salts, flavoring,coloring, and sweetening agents as appropriate. Preparations for oraladministration may be suitably formulated for slow release, controlledrelease, or sustained release of a prophylactic or therapeutic agent(s).

The method of the invention may comprise pulmonary administration, e.g.,by use of an inhaler or nebulizer, of a composition formulated with anaerosolizing agent. See, e.g., U.S. Pat. Nos. 6,019,968; 5,985,320;5,985,309; 5,934,272; 5,874,064; 5,855,913; 5,290,540; and 4,880,078;and PCT Publication Nos. WO 92/19244; WO 97/32572; WO 97/44013; WO98/31346; and WO 99/66903. In a specific embodiment, a binding proteinof the invention, combination therapy, and/or composition of theinvention is administered using Alkermes AIR® pulmonary drug deliverytechnology (Alkermes, Inc., Cambridge, Mass.).

The method of the invention may comprise administration of a compositionformulated for parenteral administration by injection (e.g., by bolusinjection or continuous infusion). Formulations for injection may bepresented in unit dosage form (e.g., in ampoules or in multi-dosecontainers) with an added preservative. The compositions may take suchforms as suspensions, solutions or emulsions in oily or aqueousvehicles, and may contain formulatory agents such as suspending,stabilizing and/or dispersing agents. Alternatively, the activeingredient may be in powder form for constitution with a suitablevehicle (e.g., sterile pyrogen-free water) before use.

The methods of the invention may additionally comprise of administrationof compositions formulated as depot preparations. Such long actingformulations may be administered by implantation (e.g., subcutaneouslyor intramuscularly) or by intramuscular injection. Thus, for example,the compositions may be formulated with suitable polymeric orhydrophobic materials (e.g., as an emulsion in an acceptable oil) or ionexchange resins, or as sparingly soluble derivatives (e.g., as asparingly soluble salt).

The methods of the invention encompass administration of compositionsformulated as neutral or salt forms. Pharmaceutically acceptable saltsinclude those formed with anions such as those derived fromhydrochloric, phosphoric, acetic, oxalic, tartaric acids, etc., andthose formed with cations such as those derived from sodium, potassium,ammonium, calcium, ferric hydroxides, isopropylamine, triethylamine,2-ethylamino ethanol, histidine, procaine, etc.

Generally, the ingredients of compositions are supplied eitherseparately or mixed together in unit dosage form, for example, as a drylyophilized powder or water free concentrate in a hermetically sealedcontainer such as an ampoule or sachette indicating the quantity ofactive agent. Where the mode of administration is infusion, compositioncan be dispensed with an infusion bottle containing sterilepharmaceutical grade water or saline. Where the mode of administrationis by injection, an ampoule of sterile water for injection or saline canbe provided so that the ingredients may be mixed prior toadministration.

In particular, the invention also provides that one or more of theprophylactic or therapeutic agents, or pharmaceutical compositions ofthe invention is packaged in a hermetically sealed container such as anampoule or sachette indicating the quantity of the agent. In oneembodiment, one or more of the prophylactic or therapeutic agents, orpharmaceutical compositions of the invention is supplied as a drysterilized lyophilized powder or water free concentrate in ahermetically sealed container and can be reconstituted (e.g., with wateror saline) to the appropriate concentration for administration to asubject. In an embodiment, one or more of the prophylactic ortherapeutic agents or pharmaceutical compositions of the invention issupplied as a dry sterile lyophilized powder in a hermetically sealedcontainer at a unit dosage of at least 5 mg, at least 10 mg, at least 15mg, at least 25 mg, at least 35 mg, at least 45 mg, at least 50 mg, atleast 75 mg, or at least 100 mg. The lyophilized prophylactic ortherapeutic agents or pharmaceutical compositions of the inventionshould be stored at between 2° C. and 8° C. in its original containerand the prophylactic or therapeutic agents, or pharmaceuticalcompositions of the invention should be administered within 1 week,e.g., within 5 days, within 72 hours, within 48 hours, within 24 hours,within 12 hours, within 6 hours, within 5 hours, within 3 hours, orwithin 1 hour after being reconstituted. In an alternative embodiment,one or more of the prophylactic or therapeutic agents or pharmaceuticalcompositions of the invention is supplied in liquid form in ahermetically sealed container indicating the quantity and concentrationof the agent. In an embodiment, the liquid form of the administeredcomposition is supplied in a hermetically sealed container at least 0.25mg/ml, at least 0.5 mg/ml, at least 1 mg/ml, at least 2.5 mg/ml, atleast 5 mg/ml, at least 8 mg/ml, at least 10 mg/ml, at least 15 mg/kg,at least 25 mg/ml, at least 50 mg/ml, at least 75 mg/ml or at least 100mg/ml. The liquid form should be stored at between 2° C. and 8° C. inits original container.

The binding proteins of the invention can be incorporated into apharmaceutical composition suitable for parenteral administration. In anembodiment, the antibody or antibody-portions will be prepared as aninjectable solution containing 0.1-250 mg/ml binding protein. Theinjectable solution can be composed of either a liquid or lyophilizeddosage form in a flint or amber vial, ampule or pre-filled syringe. Thebuffer can be L-histidine (1-50 mM), optimally 5-10 mM, at pH 5.0 to 7.0(optimally pH 6.0). Other suitable buffers include but are not limitedto, sodium succinate, sodium citrate, sodium phosphate or potassiumphosphate. Sodium chloride can be used to modify the toxicity of thesolution at a concentration of 0-300 mM (optimally 150 mM for a liquiddosage form). Cryoprotectants can be included for a lyophilized dosageform, principally 0-10% sucrose (optimally 0.5-1.0%). Other suitablecryoprotectants include trehalose and lactose. Bulking agents can beincluded for a lyophilized dosage form, principally 1-10% mannitol(optimally 2-4%). Stabilizers can be used in both liquid and lyophilizeddosage forms, principally 1-50 mM L-Methionine (optimally 5-10 mM).Other suitable bulking agents include glycine and arginine, either ofwhich can be included at a concentration of 0-0.05%, and polysorbate-80(optimally included at a concentration of 0.005-0.01%). Thepharmaceutical composition comprising the binding proteins of theinvention prepared as an injectable solution for parenteraladministration, can further comprise an agent useful as an adjuvant,such as those used to increase the absorption, or dispersion of atherapeutic protein (e.g., antibody). A particularly useful adjuvant ishyaluronidase, such as Hylenex® (recombinant human hyaluronidase).Addition of hyaluronidase in the injectable solution improves humanbioavailability following parenteral administration, particularlysubcutaneous administration. It also allows for greater injection sitevolumes (i.e., greater than 1 ml) with less pain and discomfort, andminimum incidence of injection site reactions. (see PCT Publication No.WO2004078140 and US Patent Application No. 2006104968).

The compositions of this invention may be in a variety of forms. Theseinclude, for example, liquid, semi-solid and solid dosage forms, such asliquid solutions (e.g., injectable and infusible solutions), dispersionsor suspensions, tablets, pills, powders, liposomes and suppositories.The form chosen depends on the intended mode of administration andtherapeutic application. Typical compositions are in the form ofinjectable or infusible solutions, such as compositions similar to thoseused for passive immunization of humans with other antibodies. Thechosen mode of administration is parenteral (e.g., intravenous,subcutaneous, intraperitoneal, intramuscular). In an embodiment, theantibody is administered by intravenous infusion or injection. Inanother embodiment, the antibody is administered by intramuscular orsubcutaneous injection.

Therapeutic compositions typically must be sterile and stable under theconditions of manufacture and storage. The composition can be formulatedas a solution, microemulsion, dispersion, liposome, or other orderedstructure suitable to high drug concentration. Sterile injectablesolutions can be prepared by incorporating the active compound (i.e.,antibody or antibody portion) in the required amount in an appropriatesolvent with one or a combination of ingredients enumerated herein, asrequired, followed by filtered sterilization. Generally, dispersions areprepared by incorporating the active compound into a sterile vehiclethat contains a basic dispersion medium and the required otheringredients from those enumerated herein. In the case of sterile,lyophilized powders for the preparation of sterile injectable solutions,the methods of preparation are vacuum drying and spray-drying thatyields a powder of the active ingredient plus any additional desiredingredient from a previously sterile-filtered solution thereof. Theproper fluidity of a solution can be maintained, for example, by the useof a coating such as lecithin, by the maintenance of the requiredparticle size in the case of dispersion and by the use of surfactants.Prolonged absorption of injectable compositions can be brought about byincluding, in the composition, an agent that delays absorption, forexample, monostearate salts and gelatin.

The binding proteins of the present invention can be administered by avariety of methods known in the art, although for many therapeuticapplications, in an embodiment, the route/mode of administration issubcutaneous injection, intravenous injection or infusion. As will beappreciated by the skilled artisan, the route and/or mode ofadministration will vary depending upon the desired results. In certainembodiments, the active compound may be prepared with a carrier thatwill protect the compound against rapid release, such as a controlledrelease formulation, including implants, transdermal patches, andmicroencapsulated delivery systems. Biodegradable, biocompatiblepolymers can be used, such as ethylene vinyl acetate, polyanhydrides,polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Manymethods for the preparation of such formulations are patented orgenerally known to those skilled in the art. See, e.g., Sustained andControlled Release Drug Delivery Systems, J. R. Robinson, ed., MarcelDekker, Inc., New York, 1978.

In certain embodiments, a binding protein of the invention may be orallyadministered, for example, with an inert diluent or an assimilableedible carrier. The compound (and other ingredients, if desired) mayalso be enclosed in a hard or soft shell gelatin capsule, compressedinto tablets, or incorporated directly into the subject's diet. For oraltherapeutic administration, the compounds may be incorporated withexcipients and used in the form of ingestible tablets, buccal tablets,troches, capsules, elixirs, suspensions, syrups, wafers, and the like.To administer a compound of the invention by other than parenteraladministration, it may be necessary to coat the compound with, orco-administer the compound with, a material to prevent its inactivation.

Supplementary active compounds can also be incorporated into thecompositions. In certain embodiments, a binding protein of the inventionis coformulated with and/or coadministered with one or more additionaltherapeutic agents that are useful for treating disorders with bindingprotein of the invention. For example, a binding protein of theinvention may be coformulated and/or coadministered with one or moreadditional antibodies that bind other targets (e.g., antibodies thatbind other cytokines or that bind cell surface molecules). Furthermore,one or more antibodies of the invention may be used in combination withtwo or more of the foregoing therapeutic agents. Such combinationtherapies may advantageously utilize lower dosages of the administeredtherapeutic agents, thus avoiding possible toxicities or complicationsassociated with the various monotherapies.

In certain embodiments, a binding protein is linked to a half-lifeextending vehicle known in the art. Such vehicles include, but are notlimited to, the Fc domain, polyethylene glycol, and dextran. Suchvehicles are described, e.g., in U.S. Pat. No. 6,660,843 and PCTPublication No. WO 99/25044.

In a specific embodiment, nucleic acid sequences encoding a bindingprotein of the invention or another prophylactic or therapeutic agent ofthe invention are administered to treat, prevent, manage, or amelioratea disorder or one or more symptoms thereof by way of gene therapy. Genetherapy refers to therapy performed by the administration to a subjectof an expressed or expressible nucleic acid. In this embodiment of theinvention, the nucleic acids produce their encoded antibody orprophylactic or therapeutic agent of the invention that mediates aprophylactic or therapeutic effect.

Any of the methods for gene therapy available in the art can be usedaccording to the present invention. For general reviews of the methodsof gene therapy, see Goldspiel et al. (1993) Clin. Pharm. 12:488-505; Wuand Wu (1991) Biother. 3:87-95; Tolstoshev (1993) Ann. Rev. Pharmacol.Toxicol. 32:573-596; Mulligan (1993) Science 260:926-932; and Morgan andAnderson (1993) Ann. Rev. Biochem. 62:191-217; May (1993) TIBTECH11(5):155-215. Methods commonly known in the art of recombinant DNAtechnology which can be used are described in Ausubel et al. (eds.),Current Protocols in Molecular Biology, John Wiley &Sons, NY (1993); andKriegler, Gene Transfer and Expression, A Laboratory Manual, StocktonPress, NY (1990). A detailed description of various methods of genetherapy are disclosed in US20090297514.

The binding proteins of the invention are useful in treating variousdiseases wherein the targets that are recognized by the binding proteinsare detrimental. Such diseases include, but are not limited to,rheumatoid arthritis, osteoarthritis, juvenile chronic arthritis, septicarthritis, Lyme arthritis, psoriatic arthritis, reactive arthritis,spondyloarthropathy, systemic lupus erythematosus, Crohn's disease,ulcerative colitis, inflammatory bowel disease, insulin dependentdiabetes mellitus, thyroiditis, asthma, allergic diseases, psoriasis,dermatitis scleroderma, graft versus host disease, organ transplantrejection, acute or chronic immune disease associated with organtransplantation, sarcoidosis, atherosclerosis, disseminatedintravascular coagulation, Kawasaki's disease, Grave's disease,nephrotic syndrome, chronic fatigue syndrome, Wegener's granulomatosis,Henoch-Schoenlein purpurea, microscopic vasculitis of the kidneys,chronic active hepatitis, uveitis, septic shock, toxic shock syndrome,sepsis syndrome, cachexia, infectious diseases, parasitic diseases,acquired immunodeficiency syndrome, acute transverse myelitis,Huntington's chorea, Parkinson's disease, Alzheimer's disease, stroke,primary biliary cirrhosis, hemolytic anemia, malignancies, heartfailure, myocardial infarction, Addison's disease, sporadic,polyglandular deficiency type I and polyglandular deficiency type II,Schmidt's syndrome, adult (acute) respiratory distress syndrome,alopecia, alopecia greata, seronegative arthopathy, arthropathy,Reiter's disease, psoriatic arthropathy, ulcerative colitic arthropathy,enteropathic synovitis, chlamydia, yersinia and salmonella associatedarthropathy, spondyloarthopathy, atheromatous disease/arteriosclerosis,atopic allergy, autoimmune bullous disease, pemphigus vulgaris,pemphigus foliaceus, pemphigoid, linear IgA disease, autoimmunehaemolytic anaemia, Coombs positive haemolytic anaemia, acquiredpernicious anaemia, juvenile pernicious anaemia, myalgicencephalitis/Royal Free Disease, chronic mucocutaneous candidiasis,giant cell arteritis, primary sclerosing hepatitis, cryptogenicautoimmune hepatitis, Acquired Immunodeficiency Disease Syndrome,Acquired Immunodeficiency Related Diseases, Hepatitis B, Hepatitis C,common varied immunodeficiency (common variable hypogammaglobulinaemia),dilated cardiomyopathy, female infertility, ovarian failure, prematureovarian failure, fibrotic lung disease, cryptogenic fibrosingalveolitis, post-inflammatory interstitial lung disease, interstitialpneumonitis, connective tissue disease associated interstitial lungdisease, mixed connective tissue disease associated lung disease,systemic sclerosis associated interstitial lung disease, rheumatoidarthritis associated interstitial lung disease, systemic lupuserythematosus associated lung disease, dermatomyositis/polymyositisassociated lung disease, Sjögren's disease associated lung disease,ankylosing spondylitis associated lung disease, vasculitic diffuse lungdisease, haemosiderosis associated lung disease, drug-inducedinterstitial lung disease, fibrosis, radiation fibrosis, bronchiolitisobliterans, chronic eosinophilic pneumonia, lymphocytic infiltrativelung disease, postinfectious interstitial lung disease, gouty arthritis,autoimmune hepatitis, type-1 autoimmune hepatitis (classical autoimmuneor lupoid hepatitis), type-2 autoimmune hepatitis (anti-LKM antibodyhepatitis), autoimmune mediated hypoglycaemia, type B insulin resistancewith acanthosis nigricans, hypoparathyroidism, acute immune diseaseassociated with organ transplantation, chronic immune disease associatedwith organ transplantation, osteoarthrosis, primary sclerosingcholangitis, psoriasis type 1, psoriasis type 2, idiopathic leucopaenia,autoimmune neutropaenia, renal disease NOS, glomerulonephritides,microscopic vasulitis of the kidneys, lyme disease, discoid lupuserythematosus, male infertility idiopathic or NOS, sperm autoimmunity,multiple sclerosis (all subtypes), sympathetic ophthalmia, pulmonaryhypertension secondary to connective tissue disease, Goodpasture'ssyndrome, pulmonary manifestation of polyarteritis nodosa, acuterheumatic fever, rheumatoid spondylitis, Still's disease, systemicsclerosis, Sjörgren's syndrome, Takayasu's disease/arteritis, autoimmunethrombocytopaenia, idiopathic thrombocytopaenia, autoimmune thyroiddisease, hyperthyroidism, goitrous autoimmune hypothyroidism(Hashimoto's disease), atrophic autoimmune hypothyroidism, primarymyxoedema, phacogenic uveitis, primary vasculitis, vitiligo acute liverdisease, chronic liver diseases, alcoholic cirrhosis, alcohol-inducedliver injury, choleosatatis, idiosyncratic liver disease, Drug-Inducedhepatitis, Non-alcoholic Steatohepatitis, allergy and asthma, group Bstreptococci (GBS) infection, mental disorders (e.g., depression andschizophrenia), Th2 Type and Th1 Type mediated diseases, acute andchronic pain (different forms of pain), and cancers such as lung,breast, stomach, bladder, colon, pancreas, ovarian, prostate and rectalcancer and hematopoietic malignancies (leukemia and lymphoma),Abetalipoprotemia, Acrocyanosis, acute and chronic parasitic orinfectious processes, acute leukemia, acute lymphoblastic leukemia(ALL), acute myeloid leukemia (AML), acute or chronic bacterialinfection, acute pancreatitis, acute renal failure, adenocarcinomas,aerial ectopic beats, AIDS dementia complex, alcohol-induced hepatitis,allergic conjunctivitis, allergic contact dermatitis, allergic rhinitis,allograft rejection, alpha-1-antitrypsin deficiency, amyotrophic lateralsclerosis, anemia, angina pectoris, anterior horn cell degeneration,anti cd3 therapy, antiphospholipid syndrome, anti-receptorhypersensitivity reactions, aordic and peripheral aneuryisms, aorticdissection, arterial hypertension, arteriosclerosis, arteriovenousfistula, ataxia, atrial fibrillation (sustained or paroxysmal), atrialflutter, atrioventricular block, B cell lymphoma, bone graft rejection,bone marrow transplant (BMT) rejection, bundle branch block, Burkitt'slymphoma, Burns, cardiac arrhythmias, cardiac stun syndrome, cardiactumors, cardiomyopathy, cardiopulmonary bypass inflammation response,cartilage transplant rejection, cerebellar cortical degenerations,cerebellar disorders, chaotic or multifocal atrial tachycardia,chemotherapy associated disorders, chromic myelocytic leukemia (CML),chronic alcoholism, chronic inflammatory pathologies, chroniclymphocytic leukemia (CLL), chronic obstructive pulmonary disease(COPD), chronic salicylate intoxication, colorectal carcinoma,congestive heart failure, conjunctivitis, contact dermatitis, corpulmonale, coronary artery disease, Creutzfeldt-Jakob disease, culturenegative sepsis, cystic fibrosis, cytokine therapy associated disorders,Dementia pugilistica, demyelinating diseases, dengue hemorrhagic fever,dermatitis, dermatologic conditions, diabetes, diabetes mellitus,diabetic ateriosclerotic disease, Diffuse Lewy body disease, dilatedcongestive cardiomyopathy, disorders of the basal ganglia, Down'sSyndrome in middle age, drug-induced movement disorders induced by drugswhich block CNS dopamine receptors, drug sensitivity, eczema,encephalomyelitis, endocarditis, endocrinopathy, epiglottitis,epstein-barr virus infection, erythromelalgia, extrapyramidal andcerebellar disorders, familial hematophagocytic lymphohistiocytosis,fetal thymus implant rejection, Friedreich's ataxia, functionalperipheral arterial disorders, fungal sepsis, gas gangrene, gastriculcer, glomerular nephritis, graft rejection of any organ or tissue,gram negative sepsis, gram positive sepsis, granulomas due tointracellular organisms, hairy cell leukemia, Hallerrorden-Spatzdisease, hashimoto's thyroiditis, hay fever, heart transplant rejection,hemachromatosis, hemodialysis, hemolytic uremic syndrome/thrombolyticthrombocytopenic purpura, hemorrhage, hepatitis (A), H is bundlearrythmias, HIV infection/HIV neuropathy, Hodgkin's disease,hyperkinetic movement disorders, hypersensitity reactions,hypersensitivity pneumonitis, hypertension, hypokinetic movementdisorders, hypothalamic-pituitary-adrenal axis evaluation, idiopathicAddison's disease, idiopathic pulmonary fibrosis, antibody mediatedcytotoxicity, Asthenia, infantile spinal muscular atrophy, inflammationof the aorta, influenza a, ionizing radiation exposure,iridocyclitis/uveitis/optic neuritis, ischemia-reperfusion injury,ischemic stroke, juvenile rheumatoid arthritis, juvenile spinal muscularatrophy, Kaposi's sarcoma, kidney transplant rejection, legionella,leishmaniasis, leprosy, lesions of the corticospinal system, lipedema,liver transplant rejection, lymphederma, malaria, malignamt Lymphoma,malignant histiocytosis, malignant melanoma, meningitis,meningococcemia, metabolic/idiopathic, migraine headache, mitochondrialmulti.system disorder, mixed connective tissue disease, monoclonalgammopathy, multiple myeloma, multiple systems degenerations (MencelDejerine-Thomas Shi-Drager and Machado-Joseph), myasthenia gravis,mycobacterium avium intracellulare, mycobacterium tuberculosis,myelodyplastic syndrome, myocardial infarction, myocardial ischemicdisorders, nasopharyngeal carcinoma, neonatal chronic lung disease,nephritis, nephrosis, neurodegenerative diseases, neurogenic I muscularatrophies, neutropenic fever, non-hodgkins lymphoma, occlusion of theabdominal aorta and its branches, occulsive arterial disorders, okt3therapy, orchitis/epidydimitis, orchitis/vasectomy reversal procedures,organomegaly, osteoporosis, pancreas transplant rejection, pancreaticcarcinoma, paraneoplastic syndrome/hypercalcemia of malignancy,parathyroid transplant rejection, pelvic inflammatory disease, perennialrhinitis, pericardial disease, peripheral atherlosclerotic disease,peripheral vascular disorders, peritonitis, pernicious anemia,pneumocystis carinii pneumonia, pneumonia, POEMS syndrome(polyneuropathy, organomegaly, endocrinopathy, monoclonal gammopathy,and skin changes syndrome), post perfusion syndrome, post pump syndrome,post-MI cardiotomy syndrome, preeclampsia, Progressive supranucleoPalsy, primary pulmonary hypertension, radiation therapy, Raynaud'sphenomenon and disease, Raynoud's disease, Refsum's disease, regularnarrow QRS tachycardia, renovascular hypertension, reperfusion injury,restrictive cardiomyopathy, sarcomas, scleroderma, senile chorea, SenileDementia of Lewy body type, seronegative arthropathies, shock, sicklecell anemia, skin allograft rejection, skin changes syndrome, smallbowel transplant rejection, solid tumors, specific arrythmias, spinalataxia, spinocerebellar degenerations, streptococcal myositis,structural lesions of the cerebellum, Subacute sclerosingpanencephalitis, Syncope, syphilis of the cardiovascular system,systemic anaphalaxis, systemic inflammatory response syndrome, systemiconset juvenile rheumatoid arthritis, T-cell or FAB ALL, Telangiectasia,thromboangitis obliterans, thrombocytopenia, toxicity, transplants,trauma/hemorrhage, type III hypersensitivity reactions, type IVhypersensitivity, unstable angina, uremia, urosepsis, urticaria,valvular heart diseases, varicose veins, vasculitis, venous diseases,venous thrombosis, ventricular fibrillation, viral and fungalinfections, vital encephalitis/aseptic meningitis, vital-associatedhemaphagocytic syndrome, Wernicke-Korsakoff syndrome, Wilson's disease,xenograft rejection of any organ or tissue. (see PCT Publication Nos.WO2002097048, WO9524918, and WO00/56772A1).

The DVD-Igs of the invention may also treat one or more of the followingdiseases: Acute coronary syndromes, Acute Idiopathic Polyneuritis, AcuteInflammatory Demyelinating Polyradiculoneuropathy, Acute ischemia, AdultStill's Disease, Alopecia greata, Anaphylaxis, Anti-PhospholipidAntibody Syndrome, Aplastic anemia, Arteriosclerosis, Atopic eczema,Atopic dermatitis, Autoimmune dermatitis, Autoimmune disorder associatedwith Streptococcus infection, Autoimmune hearingloss, AutoimmuneLymphoproliferative Syndrome (ALPS), Autoimmune myocarditis, autoimmunethrombocytopenia (AITP), Blepharitis, Bronchiectasis, Bullouspemphigoid, Cardiovascular Disease, Catastrophic AntiphospholipidSyndrome, Celiac Disease, Cervical Spondylosis, Chronic ischemia,Cicatricial pemphigoid, Clinically isolated Syndrome (CIS) with Risk forMultiple Sclerosis, Conjunctivitis, Childhood Onset PsychiatricDisorder, Chronic obstructive pulmonary disease (COPD), Dacryocystitis,dermatomyositis, Diabetic retinopathy, Diabetes mellitus, Diskherniation, Disk prolaps, Drug induced immune hemolytic anemia,Endocarditis, Endometriosis, endophthalmitis, Episcleritis, Erythemamultiforme, erythema multiforme major, Gestational pemphigoid,Guillain-Barré Syndrome (GBS), Hay Fever, Hughes Syndrome, IdiopathicParkinson's Disease, idiopathic interstitial pneumonia, IgE-mediatedAllergy, Immune hemolytic anemia, Inclusion Body Myositis, Infectiousocular inflammatory disease, Inflammatory demyelinating disease,Inflammatory heart disease, Inflammatory kidney disease, IPF/UIP,Iritis, Keratitis, Keratojuntivitis sicca, Kussmaul disease orKussmaul-Meier Disease, Landry's Paralysis, Langerhan's CellHistiocytosis, Livedo reticularis, Macular Degeneration, malignancies,Microscopic Polyangiitis, Morbus Bechterev, Motor Neuron Disorders,Mucous membrane pemphigoid, Multiple Organ failure, Myasthenia Gravis,Myelodysplastic Syndrome, Myocarditis, Nerve Root Disorders, Neuropathy,Non-A Non-B Hepatitis, Optic Neuritis, Osteolysis, Ovarian cancer,Pauciarticular JRA, peripheral artery occlusive disease (PAOD),peripheral vascular disease (PVD), peripheral artery disease (PAD),Phlebitis, Polyarteritis nodosa (or periarteritis nodosa),Polychondritis, Polymyalgia Rheumatica, Poliosis, Polyarticular JRA,Polyendocrine Deficiency Syndrome, Polymyositis, polymyalgia rheumatica(PMR), Post-Pump Syndrome, primary parkinsonism, prostate and rectalcancer and hematopoietic malignancies (leukemia and lymphoma),Prostatitis, Pure red cell aplasia, Primary Adrenal Insufficiency,Recurrent Neuromyelitis Optica, Restenosis, Rheumatic heart disease,SAPHO (synovitis, acne, pustulosis, hyperostosis, and osteitis),Scleroderma, Secondary Amyloidosis, Shock lung, Scleritis, Sciatica,Secondary Adrenal Insufficiency, Silicone associated connective tissuedisease, Sneddon-Wilkinson Dermatosis, spondilitis ankylosans,Stevens-Johnson Syndrome (SJS), Systemic inflammatory response syndrome,Temporal arteritis, toxoplasmic retinitis, toxic epidermal necrolysis,Transverse myelitis, TRAPS (Tumor Necrosis Factor Receptor, Type 1allergic reaction, Type II Diabetes, Urticaria, Usual interstitialpneumonia (UIP), Vasculitis, Vernal conjunctivitis, viral retinitis,Vogt-Koyanagi-Harada syndrome (VKH syndrome), Wet macular degeneration,and Wound healing.

The binding proteins of the invention can be used to treat humanssuffering from autoimmune diseases, in particular those associated withinflammation, including, rheumatoid arthritis, spondylitis, allergy,autoimmune diabetes, autoimmune uveitis. In an embodiment, the bindingproteins of the invention or antigen-binding portions thereof, are usedto treat rheumatoid arthritis, Crohn's disease, multiple sclerosis,insulin dependent diabetes mellitus and psoriasis.

In an embodiment, diseases that can be treated or diagnosed with thecompositions and methods of the invention include, but are not limitedto, primary and metastatic cancers, including carcinomas of breast,colon, rectum, lung, oropharynx, hypopharynx, esophagus, stomach,pancreas, liver, gallbladder and bile ducts, small intestine, urinarytract (including kidney, bladder and urothelium), female genital tract(including cervix, uterus, and ovaries as well as choriocarcinoma andgestational trophoblastic disease), male genital tract (includingprostate, seminal vesicles, testes and germ cell tumors), endocrineglands (including the thyroid, adrenal, and pituitary glands), and skin,as well as hemangiomas, melanomas, sarcomas (including those arisingfrom bone and soft tissues as well as Kaposi's sarcoma), tumors of thebrain, nerves, eyes, and meninges (including astrocytomas, gliomas,glioblastomas, retinoblastomas, neuromas, neuroblastomas, Schwannomas,and meningiomas), solid tumors arising from hematopoietic malignanciessuch as leukemias, and lymphomas (both Hodgkin's and non-Hodgkin'slymphomas).

In an embodiment, the antibodies of the invention or antigen-bindingportions thereof, are used to treat cancer or in the prevention orinhibition of metastases from the tumors described herein either whenused alone or in combination with radiotherapy and/or otherchemotherapeutic agents.

In another embodiment, a DVD-Ig of the invention binds a prophylactic ortherapeutic agent and a cellular protein, thereby providing forlocalized drug delivery to a specific target organ, tissue or cell, orclass of tissues or cells. In an embodiment, the DVD-Ig binds to a cellsurface antigen and a prophylactic or therapeutic agent. Theprophylactic agent or therapeutic agent is useful for preventing,managing, treating, or ameliorating a disorder or one or more symptomsthereof, e.g., liposomal particles, microparticles, microcapsules,recombinant cells capable of expressing the antibody or antibodyfragment, stem cells, receptor-mediated endocytosis (see, e.g., Wu andWu (1987) J. Biol. Chem. 262:4429-4432), peptide, nucleic acid (e.g.,antisense DND or RNA or other genetic therapy), peptide nucleic acid(PNA), nanoparticle, radiotherapeutic agent, retroviral or other vector,antibacterial, anti-viral, anti-parasitic, or antifungal agent,anti-neoplastic agents, chemotherapeutic agent, such as DNA alkylatingagents, cisplatin, carboplatin, anti-tubulin agents, paclitaxel,docetaxel, taxol, doxorubicin, gemcitabine, gemzar, anthracyclines,adriamycin, topoisomerase I inhibitors, topoisomerase II inhibitors,5-fluorouracil (5-FU), leucovorin, irinotecan, receptor tyrosine kinaseinhibitors (e.g., erlotinib, gefitinib), COX-2 inhibitors (e.g.,celecoxib), kinase inhibitors, and siRNAs, cytokine suppressiveanti-inflammatory drug(s) (CSAIDs).

In an embodiment, the DVD-Igs of the invention bind to methotrexate,6-MP, azathioprine sulphasalazine, mesalazine, olsalazinechloroquinine/hydroxychloroquine, pencillamine, aurothiomalate,azathioprine, cochicine, corticosteroids, beta-2 adrenoreceptor agonists(salbutamol, terbutaline, salmeteral), xanthines (theophylline,aminophylline), cromoglycate, nedocromil, ketotifen, ipratropium andoxitropium, cyclosporin, FK506, rapamycin, mycophenolate mofetil,leflunomide, NSAIDs, for example, ibuprofen, corticosteroids such asprednisolone, phosphodiesterase inhibitors, adensosine agonists,antithrombotic agents, complement inhibitors, adrenergic agents, agentswhich interfere with signalling by proinflammatory cytokines such asTNF-α or IL-1 (e.g., IRAK, NIK, IKK, p38 or MAP kinase inhibitors),IL-1b converting enzyme inhibitors, TNFá converting enzyme (TACE)inhibitors, T-cell signalling inhibitors such as kinase inhibitors,metalloproteinase inhibitors, sulfasalazine, azathioprine,6-mercaptopurines, angiotensin converting enzyme inhibitors, solublecytokine receptors and derivatives thereof (e.g., soluble p55 or p75 TNFreceptors and the derivatives p75TNFRIgG (Enbrel™ and p55TNFRIgG(Lenercept)), sIL-1RI, sIL-1RII, sIL-6R), growth factors, cytokines,cytotoxi proteins (e.g., TNF), antiinflammatory cytokines (e.g., IL-4,IL-10, IL-11, IL-13 and TGFβ), celecoxib, folic acid, hydroxychloroquinesulfate, rofecoxib, antibodies or a derivative or conjugate thereof(e.g., infliximab or rituximab), naproxen, valdecoxib, sulfasalazine,methylprednisolone, meloxicam, methylprednisolone acetate, gold sodiumthiomalate, aspirin, triamcinolone acetonide, propoxyphenenapsylate/apap, folate, nabumetone, diclofenac, piroxicam, etodolac,diclofenac sodium, oxaprozin, oxycodone hcl, hydrocodonebitartrate/apap, diclofenac sodium/misoprostol, fentanyl, anakinra,human recombinant, tramadol hcl, salsalate, sulindac,cyanocobalamin/fa/pyridoxine, acetaminophen, alendronate sodium,prednisolone, morphine sulfate, lidocaine hydrochloride, indomethacin,glucosamine sulf/chondroitin, amitriptyline hcl, sulfadiazine, oxycodonehcl/acetaminophen, olopatadine hcl, misoprostol, naproxen sodium,omeprazole, cyclophosphamide, rituximab, IL-1 TRAP, MRA, CTLA4-IG, IL-18BP, anti-IL-18, Anti-IL15, BIRB-796, SCIO-469, VX-702, AMG-548, VX-740,Roflumilast, IC-485, CDC-801, and Mesopram.

In another embodiment, the DVD-Ig of the invention binds tonon-steroidal anti-inflammatory drug(s) (NSAIDs); cytokine suppressiveanti-inflammatory drug(s) (CSAIDs); antibodies or derivatives orconjugates thereof [e.g., CDP-571/BAY-10-3356 (humanized anti-TNFaantibody; Celltech/Bayer); cA2/infliximab (chimeric anti-TNFa antibody;Centocor); 75 kdTNFR-IgG/etanercept (75 kD TNF receptor-IgG fusionprotein; Immunex); 55 kdTNF-IgG (55 kD TNF receptor-IgG fusion protein;Hoffmann-LaRoche); IDEC-CE9.1/SB 210396 (non-depleting primatizedanti-CD4 antibody; IDEC/SmithKline; DAB 486-IL-2 and/or DAB 389-IL-2(IL-2 fusion proteins; Seragen); Anti-Tac (humanized anti-IL-2Ra;Protein Design Labs/Roche)]; IL-4 (anti-inflammatory cytokine;DNAX/Schering); IL-10 (SCH 52000; recombinant IL-10, anti-inflammatorycytokine; DNAX/Schering); IL-4; IL-10 and/or IL-4 agonists (e.g.,agonist antibodies); IL-1RA (IL-1 receptor antagonist; Synergen/Amgen);anakinra (Kineret®/Amgen); TNF-bp/s-TNF (soluble TNF binding protein);R973401 (phosphodiesterase Type IV inhibitor); MK-966 (COX-2 Inhibitor);Iloprost; methotrexate; thalidomide and thalidomide-related drugs (e.g.,Celgen); leflunomide (anti-inflammatory and cytokine inhibitor);tranexamic acid (inhibitor of plasminogen activation); T-614 (cytokineinhibitor); prostaglandin E1); Tenidap (non-steroidal anti-inflammatorydrug); Naproxen (non-steroidal anti-inflammatory drug); Meloxicam(non-steroidal anti-inflammatory drug); Ibuprofen (non-steroidalanti-inflammatory drug); Piroxicam (non-steroidal anti-inflammatorydrug); Diclofenac (non-steroidal anti-inflammatory drug); Indomethacin(non-steroidal anti-inflammatory drug); Sulfasalazine; Azathioprine);ICE inhibitor (inhibitor of the enzyme interleukin-1b convertingenzyme); zap-70 and/or lck inhibitor (inhibitor of the tyrosine kinasezap-70 or lck); VEGF inhibitor and/or VEGF-R inhibitor (inhibitors ofvascular endothelial cell growth factor or vascular endothelial cellgrowth factor receptor; inhibitors of angiogenesis); corticosteroidanti-inflammatory drugs (e.g., SB203580); TNF-convertase inhibitors;anti-IL-12 or anti-IL-18 antibodies or derivatives or conjugatesthereof; interleukin-11; interleukin-13; interleukin-17 inhibitors;gold; penicillamine; chloroquine; chlorambucil; hydroxychloroquine;cyclosporine; cyclophosphamide; total lymphoid irradiation;anti-thymocyte globulin or anti-CD4 antibodies or derivates orconjugates thereof; CD5-toxins; orally-administered peptides andcollagen; lobenzarit disodium; Cytokine Regulating Agents (CRAs) HP228and HP466 (Houghten Pharmaceuticals, Inc.); ICAM-1 antisensephosphorothioate oligo-deoxynucleotides (ISIS 2302; IsisPharmaceuticals, Inc.); soluble complement receptor 1 (TP10; T CellSciences, Inc.); prednisone; orgotein; glycosaminoglycan polysulphate;minocycline; anti-IL2R antibodies or derivates or conjugates thereof;marine and botanical lipids (fish and plant seed fatty acids; see, e.g.,DeLuca et al. (1995) Rheum. Dis. Clin. North Am. 21:759-777); auranofin;phenylbutazone; meclofenamic acid; flufenamic acid; intravenous immuneglobulin; zileuton; azaribine; mycophenolic acid (RS-61443); tacrolimus(FK-506); sirolimus (rapamycin); amiprilose (therafectin); cladribine(2-chlorodeoxyadenosine); methotrexate; bcl-2 inhibitors (see Bruncko etal. (2007) J. Med. Chem. 50(4):641-662); antivirals and immunemodulating agents.

In one embodiment, the DVD-Ig of the invention binds to one of thefollowing agents for the treatment of rheumatoid arthritis, for example,small molecule inhibitor of KDR, small molecule inhibitor of Tie-2;methotrexate; prednisone; celecoxib; folic acid; hydroxychloroquinesulfate; rofecoxib; etanercept or infliximab or derivates or conjugatesthereof; leflunomide; naproxen; valdecoxib; sulfasalazine;methylprednisolone; ibuprofen; meloxicam; methylprednisolone acetate;gold sodium thiomalate; aspirin; azathioprine; triamcinolone acetonide;propxyphene napsylate/apap; folate; nabumetone; diclofenac; piroxicam;etodolac; diclofenac sodium; oxaprozin; oxycodone hcl; hydrocodonebitartrate/apap; diclofenac sodium/misoprostol; fentanyl; anakinra,human recombinant; tramadol hcl; salsalate; sulindac;cyanocobalamin/fa/pyridoxine; acetaminophen; alendronate sodium;prednisolone; morphine sulfate; lidocaine hydrochloride; indomethacin;glucosamine sulfate/chondroitin; cyclosporine; amitriptyline hcl;sulfadiazine; oxycodone hcl/acetaminophen; olopatadine hcl; misoprostol;naproxen sodium; omeprazole; mycophenolate mofetil; cyclophosphamide;rituximab or derivates or conjugates thereof; IL-1 TRAP; MRA; CTLA4-Igor derivates or conjugates thereof; IL-18 BP; IL-12/23; anti-IL 18 orderivates or conjugates thereof; anti-IL 15 or derivates or conjugatesthereof; BIRB-796; SC10-469; VX-702; AMG-548; VX-740; Roflumilast;IC-485; CDC-801; and mesopram.

In another embodiment, the DVD-Ig of the invention binds to therapeuticagents for inflammatory bowel disease, for example, budenoside;epidermal growth factor; corticosteroids; cyclosporin, sulfasalazine;aminosalicylates; 6-mercaptopurine; azathioprine; metronidazole;lipoxygenase inhibitors; mesalamine; olsalazine; balsalazide;antioxidants; thromboxane inhibitors; IL-1 receptor antagonists;anti-IL-1b mAbs or derivates or conjugates thereof; anti-IL-6 mAbs orderivates or conjugates thereof; growth factors; elastase inhibitors;pyridinyl-imidazole compounds; antibodies to or antagonists of otherhuman cytokines or growth factors, for example, TNF, LT, IL-1, IL-2,IL-6, IL-7, IL-8, IL-15, IL-16, IL-17, IL-18, EMAP-II, GM-CSF, FGF, andPDGF or derivates or conjugates thereof.

In one embodiment, the DVD-Ig of the invention binds to cell surfacemolecules such as CD2, CD3, CD4, CD8, CD25, CD28, CD30, CD40, CD45, CD69as methotrexate, cyclosporin, FK506, rapamycin, mycophenolate mofetil,leflunomide, NSAIDs, for example, ibuprofen, corticosteroids such asprednisolone, phosphodiesterase inhibitors, adenosine agonists,antithrombotic agents, complement inhibitors, adrenergic agents, agentswhich interfere with signalling by proinflammatory cytokines such asTNFa or IL-1 (e.g., IRAK, NIK, IKK, p38 or MAP kinase inhibitors), IL-1bconverting enzyme inhibitors, TNFa converting enzyme inhibitors, T-cellsignalling inhibitors such as kinase inhibitors, metalloproteinaseinhibitors, sulfasalazine, azathioprine, 6-mercaptopurines, angiotensinconverting enzyme inhibitors, soluble cytokine receptors and derivativesthereof (e.g., soluble p55 or p75 TNF receptors, sIL-1RI, sIL-1RII,sIL-6R) and antiinflammatory cytokines (e.g., IL-4, IL-10, IL-11, IL-13and TGFb) and bcl-2 inhibitors.

In one embodiment, the DVD-Ig of the invention binds to therapeuticagents for Crohn's disease, for example, TNF antagonists, for example,anti-TNF antibodies, Adalimumab (PCT Publication No. WO 97/29131;Humira), CA2 (Remicade), CDP 571, TNFR-Ig constructs, (p75TNFRIgG(Enbrel) and p55TNFRIgG (Lenercept)) inhibitors or derivates orconjugates thereof and PDE4 inhibitors. In one embodiment, the DVD-Ig ofthe invention binds to corticosteroids, for example, budenoside anddexamethasone. In one embodiment, the DVD-Ig of the invention binds tosulfasalazine, 5-aminosalicylic acid and olsalazine, and agents whichinterfere with synthesis or action of proinflammatory cytokines such asIL-1, for example, IL-1b converting enzyme inhibitors and IL-1ra. In oneembodiment, the DVD-Ig of the invention binds to T cell signalinginhibitors, for example, tyrosine kinase inhibitors 6-mercaptopurines.In one embodiment, the DVD-Ig of the invention binds to IL-11. In oneembodiment, the DVD-Ig of the invention binds to mesalamine, prednisone,azathioprine, mercaptopurine, infliximab or derivates or conjugatesthereof, methylprednisolone sodium succinate, diphenoxylate/atropsulfate, loperamide hydrochloride, methotrexate, omeprazole, folate,ciprofloxacin/dextrose-water, hydrocodone bitartrate/apap, tetracyclinehydrochloride, fluocinonide, metronidazole, thimerosal/boric acid,cholestyramine/sucrose, ciprofloxacin hydrochloride, hyoscyaminesulfate, meperidine hydrochloride, midazolam hydrochloride, oxycodonehcl/acetaminophen, promethazine hydrochloride, sodium phosphate,sulfamethoxazole/trimethoprim, celecoxib, polycarbophil, propoxyphenenapsylate, hydrocortisone, multivitamins, balsalazide disodium, codeinephosphate/apap, colesevelam hcl, cyanocobalamin, folic acid,levofloxacin, methylprednisolone, natalizumab or derivates or conjugatesthereof and interferon-alpha, interferon-beta, and interferon-gamma.

In one embodiment, the DVD-Ig of the invention binds to therapeuticagents for multiple sclerosis, for example, corticosteroids;prednisolone; methylprednisolone; azathioprine; cyclophosphamide;cyclosporine; methotrexate; 4-aminopyridine; tizanidine; interferon-b1a(AVONEX; Biogen); interferon-b1b (BETASERON; Chiron/Berlex); interferona-n3) (Interferon Sciences/Fujimoto), interferon-a (AlfaWassermann/J&J), interferon b1A-IF (Serono/Inhale Therapeutics),Peginterferon a 2b (Enzon/Schering-Plough), Copolymer 1 (Cop-1;COPAXONE; Teva Pharmaceutical Industries, Inc.); hyperbaric oxygen;intravenous immunoglobulin; clabribine; antibodies to or antagonists ofother human cytokines or growth factors and their receptors, forexample, TNF, LT, IL-1, IL-2, IL-6, IL-7, IL-8, IL-23, IL-15, IL-16,IL-18, EMAP-II, GM-CSF, FGF, and PDGF or derivatives or conjugatesthereof. In one embodiment, the DVD-Ig of the invention binds to cellsurface molecules such as CD2, CD3, CD4, CD8, CD19, CD20, CD25, CD28,CD30, CD40, CD45, CD69, CD80, CD86, CD90 or their ligands. In oneembodiment, the DVD-Ig of the invention binds to methotrexate,cyclosporine, FK506, rapamycin, mycophenolate mofetil, leflunomide,NSAIDs, for example, ibuprofen, corticosteroids such as prednisolone,phosphodiesterase inhibitors, adensosine agonists, antithromboticagents, complement inhibitors, adrenergic agents, agents which interferewith signalling by proinflammatory cytokines such as TNFα or IL-1 (e.g.,IRAK, NIK, IKK, p38 or MAP kinase inhibitors), IL-1β converting enzymeinhibitors, TACE inhibitors, T-cell signaling inhibitors such as kinaseinhibitors, metalloproteinase inhibitors, sulfasalazine, azathioprine,6-mercaptopurines, angiotensin converting enzyme inhibitors, solublecytokine receptors and derivatives thereof (e.g., soluble p55 or p75 TNFreceptors, sIL-1RI, sIL-1RII, sIL-6R), antiinflammatory cytokines (e.g.,IL-4, IL-10, IL-13 and TGFβ) and bcl-2 inhibitors.

In another embodiment, the DVD-Ig of the invention binds to therapeuticagents for multiple sclerosis, for example, interferon-b, for example,IFNb1a and IFNb1b; copaxone, corticosteroids, caspase inhibitors, forexample inhibitors of caspase-1, IL-1 inhibitors, TNF inhibitors, andantibodies to CD40 and CD80, and derivates or conjugates thereof.

In another embodiment, the DVD-Ig of the invention binds to thefollowing agents or derivatives or conjugates thereof: alemtuzumab,dronabinol, Unimed, daclizumab, mitoxantrone, xaliproden hydrochloride,fampridine, glatiramer acetate, natalizumab, sinnabidol, a-immunokineNNSO3, ABR-215062, AnergiX.MS, chemokine receptor antagonists, BBR-2778,calagualine, CPI-1189, LEM (liposome encapsulated mitoxantrone), THC.CBD(cannabinoid agonist) MBP-8298, mesopram (PDE4 inhibitor), MNA-715,anti-IL-6 receptor antibody, neurovax, pirfenidone allotrap 1258(RDP-1258), sTNF-R1, talampanel, teriflunomide, TGF-beta2, tiplimotide,VLA-4 antagonists (for example, TR-14035, VLA4 Ultrahaler,Antegran-ELAN/Biogen), interferon gamma antagonists, IL-4 agonists.

In another embodiment, the DVD-Ig of the invention binds to therapeuticagents for Angina, for example, nitroglycerin, isosorbide mononitrate,metoprolol succinate, atenolol, metoprolol tartrate, amlodipinebesylate, diltiazem hydrochloride, isosorbide dinitrate, clopidogrelbisulfate, nifedipine, atorvastatin calcium, potassium chloride,furosemide, simvastatin, verapamil hcl, digoxin, propranololhydrochloride, carvedilol, lisinopril, spironolactone,hydrochlorothiazide, enalapril maleate, nadolol, ramipril, enoxaparinsodium, heparin sodium, valsartan, sotalol hydrochloride, fenofibrate,ezetimibe, bumetanide, losartan potassium,lisinopril/hydrochlorothiazide, felodipine, captopril, bisoprololfumarate.

In another embodiment, the DVD-Ig of the invention binds to therapeuticagents for Ankylosing Spondylitis, for example, ibuprofen, diclofenacand misoprostol, naproxen, meloxicam, indomethacin, diclofenac,celecoxib, rofecoxib, Sulfasalazine, Methotrexate, azathioprine,minocyclin, prednisone, etanercept, infliximab, and derivatives orconjugates thereof.

In another embodiment, the DVD-Ig of the invention binds to therapeuticagents for Asthma, for example, albuterol, salmeterol/fluticasone,montelukast sodium, fluticasone propionate, budesonide, prednisone,salmeterol xinafoate, levalbuterol hcl, albuterol sulfate/ipratropium,prednisolone sodium phosphate, triamcinolone acetonide, beclomethasonedipropionate, ipratropium bromide, azithromycin, pirbuterol acetate,prednisolone, theophylline anhydrous, methylprednisolone sodiumsuccinate, clarithromycin, zafirlukast, formoterol fumarate, influenzavirus vaccine, methylprednisolone, amoxicillin trihydrate, flunisolide,allergy injection, cromolyn sodium, fexofenadine hydrochloride,flunisolide/menthol, amoxicillin/clavulanate, levofloxacin, inhalerassist device, guaifenesin, dexamethasone sodium phosphate, moxifloxacinhcl, doxycycline hyclate, guaifenesin/d-methorphan,p-ephedrine/cod/chlorphenir, gatifloxacin, cetirizine hydrochloride,mometasone furoate, salmeterol xinafoate, benzonatate, cephalexin,pe/hydrocodone/chlorphenir, cetirizine hcl/pseudoephed,phenylephrine/cod/promethazine, codeine/promethazine, cefprozil,dexamethasone, guaifenesin/pseudoephedrine,chlorpheniramine/hydrocodone, nedocromil sodium, terbutaline sulfate,epinephrine, methylprednisolone, metaproterenol sulfate.

In another embodiment, the DVD-Ig of the invention binds to therapeuticagents for COPD, for example, albuterol sulfate/ipratropium, ipratropiumbromide, salmeterol/fluticasone, albuterol, salmeterol xinafoate,fluticasone propionate, prednisone, theophylline anhydrous,methylprednisolone sodium succinate, montelukast sodium, budesonide,formoterol fumarate, triamcinolone acetonide, levofloxacin, guaifenesin,azithromycin, beclomethasone dipropionate, levalbuterol hcl,flunisolide, ceftriaxone sodium, amoxicillin trihydrate, gatifloxacin,zafirlukast, amoxicillin/clavulanate, flunisolide/menthol,chlorpheniramine/hydrocodone, metaproterenol sulfate,methylprednisolone, mometasone furoate, p-ephedrine/cod/chlorphenir,pirbuterol acetate, p-ephedrine/loratadine, terbutaline sulfate,tiotropium bromide, (R,R)-formoterol, TgAAT, Cilomilast, Roflumilast.

In another embodiment, the DVD-Ig of the invention binds to therapeuticagents for HCV, for example, Interferon-alpha-2a, Interferon-alpha-2b,Interferon-alpha con1, Interferon-alpha-n1, Pegylatedinterferon-alpha-2a, Pegylated interferon-alpha-2b, ribavirin,Peginterferon alfa-2b+ribavirin, Ursodeoxycholic Acid, GlycyrrhizicAcid, Thymalfasin, Maxamine, VX-497 and any compounds that are used totreat HCV through intervention with the following targets: HCVpolymerase, HCV protease, HCV helicase, HCV IRES (internal ribosomeentry site).

In another embodiment, the DVD-Ig of the invention binds to therapeuticagents for Idiopathic Pulmonary Fibrosis, for example, prednisone,azathioprine, albuterol, colchicine, albuterol sulfate, digoxin, gammainterferon, methylprednisolone sod succ, lorazepam, furosemide,lisinopril, nitroglycerin, spironolactone, cyclophosphamide, ipratropiumbromide, actinomycin d, alteplase, fluticasone propionate, levofloxacin,metaproterenol sulfate, morphine sulfate, oxycodone hcl, potassiumchloride, triamcinolone acetonide, tacrolimus anhydrous, calcium,interferon-alpha, methotrexate, mycophenolate mofetil,Interferon-gamma-1â.

In another embodiment, the DVD-Ig of the invention binds to therapeuticagents for Myocardial Infarction, for example, aspirin, nitroglycerin,metoprolol tartrate, enoxaparin sodium, heparin sodium, clopidogrelbisulfate, carvedilol, atenolol, morphine sulfate, metoprolol succinate,warfarin sodium, lisinopril, isosorbide mononitrate, digoxin,furosemide, simvastatin, ramipril, tenecteplase, enalapril maleate,torsemide, retavase, losartan potassium, quinapril hcl/mag carb,bumetanide, alteplase, enalaprilat, amiodarone hydrochloride, tirofibanhcl m-hydrate, diltiazem hydrochloride, captopril, irbesartan,valsartan, propranolol hydrochloride, fosinopril sodium, lidocainehydrochloride, eptifibatide, cefazolin sodium, atropine sulfate,aminocaproic acid, spironolactone, interferon, sotalol hydrochloride,potassium chloride, docusate sodium, dobutamine hcl, alprazolam,pravastatin sodium, atorvastatin calcium, midazolam hydrochloride,meperidine hydrochloride, isosorbide dinitrate, epinephrine, dopaminehydrochloride, bivalirudin, rosuvastatin, ezetimibe/simvastatin,avasimibe, cariporide, cardiac stem cells, and growth factors.

In another embodiment, the DVD-Ig of the invention binds to therapeuticagents for Psoriasis, for example, a small molecule inhibitor of KDR,small molecule inhibitor of Tie-2, calcipotriene, clobetasol propionate,triamcinolone acetonide, halobetasol propionate, tazarotene,methotrexate, fluocinonide, betamethasone diprop augmented, fluocinoloneacetonide, acitretin, tar shampoo, betamethasone valerate, mometasonefuroate, ketoconazole, pramoxine/fluocinolone, hydrocortisone valerate,flurandrenolide, urea, betamethasone, clobetasol propionate/emoll,fluticasone propionate, azithromycin, hydrocortisone, moisturizingformula, folic acid, desonide, pimecrolimus, coal tar, diflorasonediacetate, etanercept folate, lactic acid, methoxsalen, hc/bismuthsubgal/znox/resor, methylprednisolone acetate, prednisone, sunscreen,halcinonide, salicylic acid, anthralin, clocortolone pivalate, coalextract, coal tar/salicylic acid, coal tar/salicylic acid/sulfur,desoximetasone, diazepam, emollient, fluocinonide/emollient, mineraloil/castor oil/na lact, mineral oil/peanut oil, petroleum/isopropylmyristate, psoralen, salicylic acid, soap/tribromsalan, thimerosal/boricacid, celecoxib, infliximab, cyclosporine, alefacept, efalizumab,tacrolimus, pimecrolimus, PUVA, UVB, sulfasalazine.

In another embodiment, the DVD-Ig of the invention binds to therapeuticagents for Psoriatic Arthritis, for example, methotrexate, etanercept,rofecoxib, celecoxib, folic acid, sulfasalazine, naproxen, leflunomide,methylprednisolone acetate, indomethacin, hydroxychloroquine sulfate,prednisone, sulindac, betamethasone diprop augmented, infliximab,methotrexate, folate, triamcinolone acetonide, diclofenac,dimethylsulfoxide, piroxicam, diclofenac sodium, ketoprofen, meloxicam,methylprednisolone, nabumetone, tolmetin sodium, calcipotriene,cyclosporine, diclofenac sodium/misoprostol, fluocinonide, glucosaminesulfate, gold sodium thiomalate, hydrocodone bitartrate/apap, ibuprofen,risedronate sodium, sulfadiazine, thioguanine, valdecoxib, alefacept,efalizumab and bcl-2 inhibitors, or derivatives or conjugates thereof.

In another embodiment, the DVD-Ig of the invention binds to therapeuticagents for Restenosis, for example, sirolimus, paclitaxel, everolimus,tacrolimus, Zotarolimus, acetaminophen.

In another embodiment, the DVD-Ig of the invention binds to therapeuticagents for Sciatica, for example, hydrocodone bitartrate/apap,rofecoxib, cyclobenzaprine hcl, methylprednisolone, naproxen, ibuprofen,oxycodone hcl/acetaminophen, celecoxib, valdecoxib, methylprednisoloneacetate, prednisone, codeine phosphate/apap, tramadol hcl/acetaminophen,metaxalone, meloxicam, methocarbamol, lidocaine hydrochloride,diclofenac sodium, gabapentin, dexamethasone, carisoprodol, ketorolactromethamine, indomethacin, acetaminophen, diazepam, nabumetone,oxycodone hcl, tizanidine hcl, diclofenac sodium/misoprostol,propoxyphene napsylate/apap, asa/oxycod/oxycodone ter,ibuprofen/hydrocodone bit, tramadol hcl, etodolac, propoxyphene hcl,amitriptyline hcl, carisoprodol/codeine phos/asa, morphine sulfate,multivitamins, naproxen sodium, orphenadrine citrate, temazepam.

In one embodiment, the DVD-Ig of the invention binds to agents for SLE(Lupus), for example, NSAIDS, for example, diclofenac, naproxen,ibuprofen, piroxicam, indomethacin; COX2 inhibitors, for example,Celecoxib, rofecoxib, valdecoxib; anti-malarials, for example,hydroxychloroquine; Steroids, for example, prednisone, prednisolone,budenoside, dexamethasone; cytotoxics, for example, azathioprine,cyclophosphamide, mycophenolate mofetil, methotrexate; inhibitors ofPDE4 or purine synthesis inhibitor, for example Cellcept. In oneembodiment, the DVD-Ig of the invention binds to sulfasalazine,5-aminosalicylic acid, olsalazine, Imuran and agents which interferewith synthesis, production or action of proinflammatory cytokines suchas IL-1, for example, caspase inhibitors like IL-1b converting enzymeinhibitors and IL-1ra. In one embodiment, the DVD-Ig of the inventionbinds to T cell signaling inhibitors, for example, tyrosine kinaseinhibitors; or molecules that target T cell activation molecules, forexample, CTLA-4-Ig or B7 family antibodies, or PD-1 family. In oneembodiment, the DVD-Ig of the invention binds to IL-11 or anti-cytokineantibodies, for example, fonotolizumab (anti-IFNg antibody), oranti-receptor receptor antibodies, for example, anti-IL-6 receptorantibody and antibodies to B-cell surface molecules. In one embodiment,the DVD-Ig of the invention binds to LJP 394 (abetimus), agents thatdeplete or inactivate B-cells, for example, anti-CD20 antibody, andBlyS, TNF and bcl-2 inhibitors, because bcl-2 overexpression intransgenic mice has been demonstrated to cause a lupus like phenotype(see Marquina et al. (2004) J. Immunol. 172(11):7177-7185), thereforeinhibition is expected to have therapeutic effects.

The antibodies of the invention, or antigen binding portions thereof,may be combined with agents that include but are not limited to,antineoplastic agents, radiotherapy, chemotherapy such as DNA alkylatingagents, cisplatin, carboplatin, anti-tubulin agents, paclitaxel,docetaxel, taxol, doxorubicin, gemcitabine, gemzar, anthracyclines,adriamycin, topoisomerase I inhibitors, topoisomerase II inhibitors,5-fluorouracil (5-FU), leucovorin, irinotecan, receptor tyrosine kinaseinhibitors (e.g., erlotinib, gefitinib), COX-2 inhibitors (e.g.,celecoxib), kinase inhibitors, and siRNAs.

A binding protein of the invention also can be administered with one ormore additional therapeutic agents useful in the treatment of variousdiseases.

A binding protein of the invention can be used alone or in combinationto treat such diseases. It should be understood that the bindingproteins can be used alone or in combination with an additional agent,e.g., a therapeutic agent, said additional agent being selected by theskilled artisan for its intended purpose. For example, the additionalagent can be a therapeutic agent art-recognized as being useful to treatthe disease or condition being treated by the antibody of the presentinvention. The additional agent also can be an agent that imparts abeneficial attribute to the therapeutic composition, e.g., an agent thataffects the viscosity of the composition.

It should further be understood that the combinations which are to beincluded within this invention are those combinations useful for theirintended purpose. The agents set forth below are illustrative and arenot intended to be limited. The combinations, which are part of thisinvention, can be the antibodies of the present invention and at leastone additional agent selected from the lists below. The combination canalso include more than one additional agent, e.g., two or threeadditional agents if the combination is such that the formed compositioncan perform its intended function.

Combinations to treat autoimmune and inflammatory diseases arenon-steroidal anti-inflammatory drug(s) also referred to as NSAIDS whichinclude drugs like ibuprofen. Other combinations are corticosteroidsincluding prednisolone; the well known side-effects of steroid use canbe reduced or even eliminated by tapering the steroid dose required whentreating patients in combination with the DVD Igs of this invention.Non-limiting examples of therapeutic agents for rheumatoid arthritiswith which an antibody, or antibody portion, of the invention can becombined include the following: cytokine suppressive anti-inflammatorydrug(s) (CSAIDs); antibodies to or antagonists of other human cytokinesor growth factors, for example, TNF, LT, IL-1, IL-2, IL-3, IL-4, IL-5,IL-6, IL-7, IL-8, IL-15, IL-16, IL-18, IL-21, IL-23, interferons,EMAP-II, GM-CSF, FGF, and PDGF. Binding proteins of the invention, orantigen binding portions thereof, can be combined with antibodies tocell surface molecules such as CD2, CD3, CD4, CD8, CD25, CD28, CD30,CD40, CD45, CD69, CD80 (B7.1), CD86 (B7.2), CD90, CTLA or their ligandsincluding CD154 (gp39 or CD40L).

Combinations of therapeutic agents may interfere at different points inthe autoimmune and subsequent inflammatory cascade; examples include TNFantagonists like chimeric, humanized or human TNF antibodies,Adalimumab, (PCT Publication No. WO 97/29131), CA2 (Remicade™), CDP 571,and soluble p55 or p75 TNF receptors, derivatives, thereof, (p75TNFR1gG(Enbrel™) or p55TNFR1gG (Lenercept), and also TNFα converting enzyme(TACE) inhibitors; similarly IL-1 inhibitors (Interleukin-1-convertingenzyme inhibitors, IL-1RA etc.) may be effective for the same reason.Other combinations include Interleukin 11. Yet another combinationinclude key players of the autoimmune response which may act parallelto, dependent on or in concert with IL-12 function; especially are IL-18antagonists including IL-18 antibodies or soluble IL-18 receptors, orIL-18 binding proteins. It has been shown that IL-12 and IL-18 haveoverlapping but distinct functions and a combination of antagonists toboth may be most effective. Yet another combination are non-depletinganti-CD4 inhibitors. Yet other combinations include antagonists of theco-stimulatory pathway CD80 (B7.1) or CD86 (B7.2) including antibodies,soluble receptors or antagonistic ligands.

The binding proteins of the invention may also be combined with agents,such as methotrexate, 6-MP, azathioprine sulphasalazine, mesalazine,olsalazine chloroquinine/hydroxychloroquine, pencillamine,aurothiomalate (intramuscular and oral), azathioprine, cochicine,corticosteroids (oral, inhaled and local injection), beta-2adrenoreceptor agonists (salbutamol, terbutaline, salmeteral), xanthines(theophylline, aminophylline), cromoglycate, nedocromil, ketotifen,ipratropium and oxitropium, cyclosporin, FK506, rapamycin, mycophenolatemofetil, leflunomide, NSAIDs, for example, ibuprofen, corticosteroidssuch as prednisolone, phosphodiesterase inhibitors, adensosine agonists,antithrombotic agents, complement inhibitors, adrenergic agents, agentswhich interfere with signalling by proinflammatory cytokines such asTNF-α or IL-1 (e.g., IRAK, NIK, IKK, p38 or MAP kinase inhibitors),IL-1β converting enzyme inhibitors, TNFα converting enzyme (TACE)inhibitors, T-cell signalling inhibitors such as kinase inhibitors,metalloproteinase inhibitors, sulfasalazine, azathioprine,6-mercaptopurines, angiotensin converting enzyme inhibitors, solublecytokine receptors and derivatives thereof (e.g., soluble p55 or p75 TNFreceptors and the derivatives p75TNFRIgG (Enbrel™ and p55TNFRIgG(Lenercept)), sIL-1RI, sIL-1RII, sIL-6R), antiinflammatory cytokines(e.g., IL-4, IL-10, IL-11, IL-13 and TGFβ), celecoxib, folic acid,hydroxychloroquine sulfate, rofecoxib, etanercept, infliximab, naproxen,valdecoxib, sulfasalazine, methylprednisolone, meloxicam,methylprednisolone acetate, gold sodium thiomalate, aspirin,triamcinolone acetonide, propoxyphene napsylate/apap, folate,nabumetone, diclofenac, piroxicam, etodolac, diclofenac sodium,oxaprozin, oxycodone hcl, hydrocodone bitartrate/apap, diclofenacsodium/misoprostol, fentanyl, anakinra, human recombinant, tramadol hcl,salsalate, sulindac, cyanocobalamin/fa/pyridoxine, acetaminophen,alendronate sodium, prednisolone, morphine sulfate, lidocainehydrochloride, indomethacin, glucosamine sulf/chondroitin, amitriptylinehcl, sulfadiazine, oxycodone hcl/acetaminophen, olopatadine hcl,misoprostol, naproxen sodium, omeprazole, cyclophosphamide, rituximab,IL-1 TRAP, MRA, CTLA4-IG, IL-18 BP, anti-IL-18, Anti-IL15, BIRB-796,SC10-469, VX-702, AMG-548, VX-740, Roflumilast, IC-485, CDC-801, andMesopram. Combinations include methotrexate or leflunomide and inmoderate or severe rheumatoid arthritis cases, cyclosporine.

Nonlimiting additional agents which can also be used in combination witha binding protein to treat rheumatoid arthritis include, but are notlimited to, the following: non-steroidal anti-inflammatory drug(s)(NSAIDs); cytokine suppressive anti-inflammatory drug(s) (CSAIDs);CDP-571/BAY-10-3356 (humanized anti-TNFα antibody; Celltech/Bayer);cA2/infliximab (chimeric anti-TNFα antibody; Centocor); 75kdTNFR-IgG/etanercept (75 kD TNF receptor-IgG fusion protein; Immunex;(1994) Arthritis & Rheumatism 37: S295; (1996) J. Invest. Med. 44:235A);55 kdTNF-IgG (55 kD TNF receptor-IgG fusion protein; Hoffmann-LaRoche);IDEC-CE9.1/SB 210396 (non-depleting primatized anti-CD4 antibody;IDEC/SmithKline; (1995) Arthrit. Rheum. 38: S185); DAB 486-IL-2 and/orDAB 389-IL-2 (IL-2 fusion proteins; Seragen; (1993) Arthrit. Rheum.36:1223); Anti-Tac (humanized anti-IL-2Rα; Protein Design Labs/Roche);IL-4 (anti-inflammatory cytokine; DNAX/Schering); IL-10 (SCH 52000;recombinant IL-10, anti-inflammatory cytokine; DNAX/Schering); IL-4;IL-10 and/or IL-4 agonists (e.g., agonist antibodies); IL-1RA (IL-1receptor antagonist; Synergen/Amgen); anakinra (Kineret®/Amgen);TNF-bp/s-TNF (soluble TNF binding protein; (1996) Arthrit. Rheum. 39 (9;supplement): S284; (1995) Amer. J. Physiol.—Heart and CirculatoryPhysiology 268:37-42); R973401 (phosphodiesterase Type IV inhibitor;(1996) Arthrit. Rheum. 39 (9; supplement): S282); MK-966 (COX-2Inhibitor; (1996) Arthrit. Rheum. 39 (9; supplement): S81); Iloprost((1996) Arthrit. Rheum. 39 (9; supplement): S82); methotrexate;thalidomide ((1996) Arthrit. Rheum. 39 (9; supplement): S282) andthalidomide-related drugs (e.g., Celgen); leflunomide (anti-inflammatoryand cytokine inhibitor; (1996) Arthrit. Rheum. 39 (9; supplement): S131;(1996) Inflammation Research 45:103-107); tranexamic acid (inhibitor ofplasminogen activation; (1996) Arthrit. Rheum. 39 (9; supplement):S284); T-614 (cytokine inhibitor; (1996) Arthrit. Rheum. 39 (9;supplement): S282); prostaglandin E1 ((1996) Arthrit. Rheum. 39 (9;supplement): S282); Tenidap (non-steroidal anti-inflammatory drug;(1996) Arthrit. Rheum. 39 (9; supplement): S280); Naproxen(non-steroidal anti-inflammatory drug; (1996) Neuro Report 7:1209-1213);Meloxicam (non-steroidal anti-inflammatory drug); Ibuprofen(non-steroidal anti-inflammatory drug); Piroxicam (non-steroidalanti-inflammatory drug); Diclofenac (non-steroidal anti-inflammatorydrug); Indomethacin (non-steroidal anti-inflammatory drug);Sulfasalazine ((1996) Arthrit. Rheum. 39 (9; supplement): S281);Azathioprine ((1996) Arthrit. Rheum. 39 (9; supplement): S281); ICEinhibitor (inhibitor of the enzyme interleukin-1β converting enzyme);zap-70 and/or lck inhibitor (inhibitor of the tyrosine kinase zap-70 orlck); VEGF inhibitor and/or VEGF-R inhibitor (inhibitors of vascularendothelial cell growth factor or vascular endothelial cell growthfactor receptor; inhibitors of angiogenesis); corticosteroidanti-inflammatory drugs (e.g., SB203580); TNF-convertase inhibitors;anti-IL-12 antibodies; anti-IL-18 antibodies; interleukin-11 ((1996)Arthrit. Rheum. 39 (9; supplement): S296); interleukin-13 ((1996)Arthrit. Rheum. 39 (9; supplement): S308); interleukin-17 inhibitors(see e.g., (1996) Arthrit. Rheum. 39 (9; supplement): S120); gold;penicillamine; chloroquine; chlorambucil; hydroxychloroquine;cyclosporine; cyclophosphamide; total lymphoid irradiation;anti-thymocyte globulin; anti-CD4 antibodies; CD5-toxins;orally-administered peptides and collagen; lobenzarit disodium; CytokineRegulating Agents (CRAs) HP228 and HP466 (Houghten Pharmaceuticals,Inc.); ICAM-1 antisense phosphorothioate oligo-deoxynucleotides (ISIS2302; Isis Pharmaceuticals, Inc.); soluble complement receptor 1 (TP10;T Cell Sciences, Inc.); prednisone; orgotein; glycosaminoglycanpolysulphate; minocycline; anti-IL2R antibodies; marine and botanicallipids (fish and plant seed fatty acids; DeLuca et al. (1995) Rheum.Dis. Clin. North Am. 21:759-777); auranofin; phenylbutazone;meclofenamic acid; flufenamic acid; intravenous immune globulin;zileuton; azaribine; mycophenolic acid (RS-61443); tacrolimus (FK-506);sirolimus (rapamycin); amiprilose (therafectin); cladribine(2-chlorodeoxyadenosine); methotrexate; bcl-2 inhibitors (Bruncko et al.(2007) J. Med. Chem. 50(4):641-662); antivirals and immune modulatingagents.

In one embodiment, the binding protein or antigen-binding portionthereof, is administered in combination with one of the following agentsfor the treatment of rheumatoid arthritis: small molecule inhibitor ofKDR, small molecule inhibitor of Tie-2; methotrexate; prednisone;celecoxib; folic acid; hydroxychloroquine sulfate; rofecoxib;etanercept; infliximab; leflunomide; naproxen; valdecoxib;sulfasalazine; methylprednisolone; ibuprofen; meloxicam;methylprednisolone acetate; gold sodium thiomalate; aspirin;azathioprine; triamcinolone acetonide; propxyphene napsylate/apap;folate; nabumetone; diclofenac; piroxicam; etodolac; diclofenac sodium;oxaprozin; oxycodone hcl; hydrocodone bitartrate/apap; diclofenacsodium/misoprostol; fentanyl; anakinra, human recombinant; tramadol hcl;salsalate; sulindac; cyanocobalamin/fa/pyridoxine; acetaminophen;alendronate sodium; prednisolone; morphine sulfate; lidocainehydrochloride; indomethacin; glucosamine sulfate/chondroitin;cyclosporine; amitriptyline hcl; sulfadiazine; oxycodonehcl/acetaminophen; olopatadine hcl; misoprostol; naproxen sodium;omeprazole; mycophenolate mofetil; cyclophosphamide; rituximab; IL-1TRAP; MRA; CTLA4-IG; IL-18 BP; IL-12/23; anti-IL 18; anti-IL 15;BIRB-796; SCIO-469; VX-702; AMG-548; VX-740; Roflumilast; IC-485;CDC-801; and mesopram.

Non-limiting examples of therapeutic agents for inflammatory boweldisease with which a binding protein of the invention can be combinedinclude the following: budenoside; epidermal growth factor;corticosteroids; cyclosporin, sulfasalazine; aminosalicylates;6-mercaptopurine; azathioprine; metronidazole; lipoxygenase inhibitors;mesalamine; olsalazine; balsalazide; antioxidants; thromboxaneinhibitors; IL-1 receptor antagonists; anti-IL-1β mAbs; anti-IL-6 mAbs;growth factors; elastase inhibitors; pyridinyl-imidazole compounds;antibodies to or antagonists of other human cytokines or growth factors,for example, TNF, LT, IL-1, IL-2, IL-6, IL-7, IL-8, IL-15, IL-16, IL-17,IL-18, EMAP-II, GM-CSF, FGF, and PDGF. Antibodies of the invention, orantigen binding portions thereof, can be combined with antibodies tocell surface molecules such as CD2, CD3, CD4, CD8, CD25, CD28, CD30,CD40, CD45, CD69, CD90 or their ligands. The antibodies of theinvention, or antigen binding portions thereof, may also be combinedwith agents, such as methotrexate, cyclosporin, FK506, rapamycin,mycophenolate mofetil, leflunomide, NSAIDs, for example, ibuprofen,corticosteroids such as prednisolone, phosphodiesterase inhibitors,adenosine agonists, antithrombotic agents, complement inhibitors,adrenergic agents, agents which interfere with signalling byproinflammatory cytokines such as TNFα or IL-1 (e.g., IRAK, NIK, IKK,p38 or MAP kinase inhibitors), IL-1β converting enzyme inhibitors, TNFαconverting enzyme inhibitors, T-cell signalling inhibitors such askinase inhibitors, metalloproteinase inhibitors, sulfasalazine,azathioprine, 6-mercaptopurines, angiotensin converting enzymeinhibitors, soluble cytokine receptors and derivatives thereof (e.g.,soluble p55 or p75 TNF receptors, sIL-1RI, sIL-1RII, sIL-6R) andantiinflammatory cytokines (e.g., IL-4, IL-10, IL-11, IL-13 and TGFβ)and bcl-2 inhibitors.

Examples of therapeutic agents for Crohn's disease in which a bindingprotein can be combined include the following: TNF antagonists, forexample, anti-TNF antibodies, Adalimumab (PCT Publication No. WO97/29131; HUMIRA), CA2 (REMICADE), CDP 571, TNFR-Ig constructs,(p75TNFRIgG (ENBREL) and p55TNFRIgG (LENERCEPT)) inhibitors and PDE4inhibitors. Antibodies of the invention, or antigen binding portionsthereof, can be combined with corticosteroids, for example, budenosideand dexamethasone. Binding proteins of the invention or antigen bindingportions thereof, may also be combined with agents such assulfasalazine, 5-aminosalicylic acid and olsalazine, and agents whichinterfere with synthesis or action of proinflammatory cytokines such asIL-1, for example, IL-1β converting enzyme inhibitors and IL-1ra.Antibodies of the invention or antigen binding portion thereof may alsobe used with T cell signaling inhibitors, for example, tyrosine kinaseinhibitors 6-mercaptopurines. Binding proteins of the invention, orantigen binding portions thereof, can be combined with IL-11. Bindingproteins of the invention, or antigen binding portions thereof, can becombined with mesalamine, prednisone, azathioprine, mercaptopurine,infliximab, methylprednisolone sodium succinate, diphenoxylate/atropsulfate, loperamide hydrochloride, methotrexate, omeprazole, folate,ciprofloxacin/dextrose-water, hydrocodone bitartrate/apap, tetracyclinehydrochloride, fluocinonide, metronidazole, thimerosal/boric acid,cholestyramine/sucrose, ciprofloxacin hydrochloride, hyoscyaminesulfate, meperidine hydrochloride, midazolam hydrochloride, oxycodonehcl/acetaminophen, promethazine hydrochloride, sodium phosphate,sulfamethoxazole/trimethoprim, celecoxib, polycarbophil, propoxyphenenapsylate, hydrocortisone, multivitamins, balsalazide disodium, codeinephosphate/apap, colesevelam hcl, cyanocobalamin, folic acid,levofloxacin, methylprednisolone, natalizumab and interferon-gamma.

Non-limiting examples of therapeutic agents for multiple sclerosis withwhich binding proteins of the invention can be combined include thefollowing: corticosteroids; prednisolone; methylprednisolone;azathioprine; cyclophosphamide; cyclosporine; methotrexate;4-aminopyridine; tizanidine; interferon-β1a (AVONEX; Biogen);interferon-β1b (BETASERON; Chiron/Berlex); interferon α-n3) (InterferonSciences/Fujimoto), interferon-α (Alfa Wassermann/J&J), interferonβ1A-IF (Serono/Inhale Therapeutics), Peginterferon α 2b(Enzon/Schering-Plough), Copolymer 1 (Cop-1; COPAXONE; TevaPharmaceutical Industries, Inc.); hyperbaric oxygen; intravenousimmunoglobulin; clabribine; antibodies to or antagonists of other humancytokines or growth factors and their receptors, for example, TNF, LT,IL-1, IL-2, IL-6, IL-7, IL-8, IL-23, IL-15, IL-16, IL-18, EMAP-II,GM-CSF, FGF, and PDGF. Binding proteins of the invention can be combinedwith antibodies to cell surface molecules such as CD2, CD3, CD4, CD8,CD19, CD20, CD25, CD28, CD30, CD40, CD45, CD69, CD80, CD86, CD90 ortheir ligands. Binding proteins of the invention, may also be combinedwith agents, such as methotrexate, cyclosporine, FK506, rapamycin,mycophenolate mofetil, leflunomide, NSAIDs, for example, ibuprofen,corticosteroids such as prednisolone, phosphodiesterase inhibitors,adensosine agonists, antithrombotic agents, complement inhibitors,adrenergic agents, agents which interfere with signalling byproinflammatory cytokines such as TNFα or IL-1 (e.g., IRAK, NIK, IKK,p38 or MAP kinase inhibitors), IL-1β converting enzyme inhibitors, TACEinhibitors, T-cell signaling inhibitors such as kinase inhibitors,metalloproteinase inhibitors, sulfasalazine, azathioprine,6-mercaptopurines, angiotensin converting enzyme inhibitors, solublecytokine receptors and derivatives thereof (e.g., soluble p55 or p75 TNFreceptors, sIL-1RI, sIL-1RII, sIL-6R), antiinflammatory cytokines (e.g.,IL-4, IL-10, IL-13 and TGFβ) and bcl-2 inhibitors.

Examples of therapeutic agents for multiple sclerosis in which bindingproteins of the invention can be combined include interferon-β, forexample, IFNβ1a and IFNβ1b; copaxone, corticosteroids, caspaseinhibitors, for example inhibitors of caspase-1, IL-1 inhibitors, TNFinhibitors, and antibodies to CD40 ligand and CD80.

The binding proteins of the invention, may also be combined with agents,such as alemtuzumab, dronabinol, Unimed, daclizumab, mitoxantrone,xaliproden hydrochloride, fampridine, glatiramer acetate, natalizumab,sinnabidol, a-immunokine NNSO3, ABR-215062, AnergiX.MS, chemokinereceptor antagonists, BBR-2778, calagualine, CPI-1189, LEM (liposomeencapsulated mitoxantrone), THC.CBD (cannabinoid agonist) MBP-8298,mesopram (PDE4 inhibitor), MNA-715, anti-IL-6 receptor antibody,neurovax, pirfenidone allotrap 1258 (RDP-1258), sTNF-R1, talampanel,teriflunomide, TGF-beta2, tiplimotide, VLA-4 antagonists (for example,TR-14035, VLA4 Ultrahaler, Antegran-ELAN/Biogen), interferon gammaantagonists, and IL-4 agonists.

Non-limiting examples of therapeutic agents for Angina with whichbinding proteins of the invention can be combined include the following:aspirin, nitroglycerin, isosorbide mononitrate, metoprolol succinate,atenolol, metoprolol tartrate, amlodipine besylate, diltiazemhydrochloride, isosorbide dinitrate, clopidogrel bisulfate, nifedipine,atorvastatin calcium, potassium chloride, furosemide, simvastatin,verapamil hcl, digoxin, propranolol hydrochloride, carvedilol,lisinopril, spironolactone, hydrochlorothiazide, enalapril maleate,nadolol, ramipril, enoxaparin sodium, heparin sodium, valsartan, sotalolhydrochloride, fenofibrate, ezetimibe, bumetanide, losartan potassium,lisinopril/hydrochlorothiazide, felodipine, captopril, and bisoprololfumarate.

Non-limiting examples of therapeutic agents for Ankylosing Spondylitiswith which binding proteins of the invention can be combined include thefollowing: ibuprofen, diclofenac and misoprostol, naproxen, meloxicam,indomethacin, diclofenac, celecoxib, rofecoxib, Sulfasalazine,Methotrexate, azathioprine, minocyclin, prednisone, etanercept, andinfliximab.

Non-limiting examples of therapeutic agents for Asthma with whichbinding proteins of the invention can be combined include the following:albuterol, salmeterol/fluticasone, montelukast sodium, fluticasonepropionate, budesonide, prednisone, salmeterol xinafoate, levalbuterolhcl, albuterol sulfate/ipratropium, prednisolone sodium phosphate,triamcinolone acetonide, beclomethasone dipropionate, ipratropiumbromide, azithromycin, pirbuterol acetate, prednisolone, theophyllineanhydrous, methylprednisolone sodium succinate, clarithromycin,zafirlukast, formoterol fumarate, influenza virus vaccine,methylprednisolone, amoxicillin trihydrate, flunisolide, allergyinjection, cromolyn sodium, fexofenadine hydrochloride,flunisolide/menthol, amoxicillin/clavulanate, levofloxacin, inhalerassist device, guaifenesin, dexamethasone sodium phosphate, moxifloxacinhcl, doxycycline hyclate, guaifenesin/d-methorphan,p-ephedrine/cod/chlorphenir, gatifloxacin, cetirizine hydrochloride,mometasone furoate, salmeterol xinafoate, benzonatate, cephalexin,pe/hydrocodone/chlorphenir, cetirizine hcl/pseudoephed,phenylephrine/cod/promethazine, codeine/promethazine, cefprozil,dexamethasone, guaifenesin/pseudoephedrine,chlorpheniramine/hydrocodone, nedocromil sodium, terbutaline sulfate,epinephrine, methylprednisolone, metaproterenol sulfate.

Non-limiting examples of therapeutic agents for COPD with which bindingproteins of the invention can be combined include the following:albuterol sulfate/ipratropium, ipratropium bromide,salmeterol/fluticasone, albuterol, salmeterol xinafoate, fluticasonepropionate, prednisone, theophylline anhydrous, methylprednisolonesodium succinate, montelukast sodium, budesonide, formoterol fumarate,triamcinolone acetonide, levofloxacin, guaifenesin, azithromycin,beclomethasone dipropionate, levalbuterol hcl, flunisolide, ceftriaxonesodium, amoxicillin trihydrate, gatifloxacin, zafirlukast,amoxicillin/clavulanate, flunisolide/menthol,chlorpheniramine/hydrocodone, metaproterenol sulfate,methylprednisolone, mometasone furoate, p-ephedrine/cod/chlorphenir,pirbuterol acetate, p-ephedrine/loratadine, terbutaline sulfate,tiotropium bromide, (R,R)-formoterol, TgAAT, Cilomilast, andRoflumilast.

Non-limiting examples of therapeutic agents for HCV with which bindingproteins of the invention can be combined include the following:Interferon-alpha-2a, Interferon-alpha-2b, Interferon-alpha con1,Interferon-alpha-n1, Pegylated interferon-alpha-2a, Pegylatedinterferon-alpha-2b, ribavirin, Peginterferon alfa-2b+ribavirin,Ursodeoxycholic Acid, Glycyrrhizic Acid, Thymalfasin, Maxamine, VX-497and any compounds that are used to treat HCV through intervention withthe following targets: HCV polymerase, HCV protease, HCV helicase, HCVIRES (internal ribosome entry site).

Non-limiting examples of therapeutic agents for Idiopathic PulmonaryFibrosis with which binding proteins of the invention can be combinedinclude the following: prednisone, azathioprine, albuterol, colchicine,albuterol sulfate, digoxin, gamma interferon, methylprednisolone sodsucc, lorazepam, furosemide, lisinopril, nitroglycerin, spironolactone,cyclophosphamide, ipratropium bromide, actinomycin d, alteplase,fluticasone propionate, levofloxacin, metaproterenol sulfate, morphinesulfate, oxycodone hcl, potassium chloride, triamcinolone acetonide,tacrolimus anhydrous, calcium, interferon-alpha, methotrexate,mycophenolate mofetil, Interferon-gamma-1β.

Non-limiting examples of therapeutic agents for Myocardial Infarctionwith which binding proteins of the invention can be combined include thefollowing: aspirin, nitroglycerin, metoprolol tartrate, enoxaparinsodium, heparin sodium, clopidogrel bisulfate, carvedilol, atenolol,morphine sulfate, metoprolol succinate, warfarin sodium, lisinopril,isosorbide mononitrate, digoxin, furosemide, simvastatin, ramipril,tenecteplase, enalapril maleate, torsemide, retavase, losartanpotassium, quinapril hcl/mag carb, bumetanide, alteplase, enalaprilat,amiodarone hydrochloride, tirofiban hcl m-hydrate, diltiazemhydrochloride, captopril, irbesartan, valsartan, propranololhydrochloride, fosinopril sodium, lidocaine hydrochloride, eptifibatide,cefazolin sodium, atropine sulfate, aminocaproic acid, spironolactone,interferon, sotalol hydrochloride, potassium chloride, docusate sodium,dobutamine hcl, alprazolam, pravastatin sodium, atorvastatin calcium,midazolam hydrochloride, meperidine hydrochloride, isosorbide dinitrate,epinephrine, dopamine hydrochloride, bivalirudin, rosuvastatin,ezetimibe/simvastatin, avasimibe, and cariporide.

Non-limiting examples of therapeutic agents for Psoriasis with whichbinding proteins of the invention can be combined include the following:small molecule inhibitor of KDR, small molecule inhibitor of Tie-2,calcipotriene, clobetasol propionate, triamcinolone acetonide,halobetasol propionate, tazarotene, methotrexate, fluocinonide,betamethasone diprop augmented, fluocinolone acetonide, acitretin, tarshampoo, betamethasone valerate, mometasone furoate, ketoconazole,pramoxine/fluocinolone, hydrocortisone valerate, flurandrenolide, urea,betamethasone, clobetasol propionate/emoll, fluticasone propionate,azithromycin, hydrocortisone, moisturizing formula, folic acid,desonide, pimecrolimus, coal tar, diflorasone diacetate, etanerceptfolate, lactic acid, methoxsalen, hc/bismuth subgal/znox/resor,methylprednisolone acetate, prednisone, sunscreen, halcinonide,salicylic acid, anthralin, clocortolone pivalate, coal extract, coaltar/salicylic acid, coal tar/salicylic acid/sulfur, desoximetasone,diazepam, emollient, fluocinonide/emollient, mineral oil/castor oil/nalact, mineral oil/peanut oil, petroleum/isopropyl myristate, psoralen,salicylic acid, soap/tribromsalan, thimerosal/boric acid, celecoxib,infliximab, cyclosporine, alefacept, efalizumab, tacrolimus,pimecrolimus, PUVA, UVB, and sulfasalazine.

Non-limiting examples of therapeutic agents for Psoriatic Arthritis withwhich binding proteins of the invention can be combined include thefollowing: methotrexate, etanercept, rofecoxib, celecoxib, folic acid,sulfasalazine, naproxen, leflunomide, methylprednisolone acetate,indomethacin, hydroxychloroquine sulfate, prednisone, sulindac,betamethasone diprop augmented, infliximab, methotrexate, folate,triamcinolone acetonide, diclofenac, dimethylsulfoxide, piroxicam,diclofenac sodium, ketoprofen, meloxicam, methylprednisolone,nabumetone, tolmetin sodium, calcipotriene, cyclosporine, diclofenacsodium/misoprostol, fluocinonide, glucosamine sulfate, gold sodiumthiomalate, hydrocodone bitartrate/apap, ibuprofen, risedronate sodium,sulfadiazine, thioguanine, valdecoxib, alefacept, efalizumab and bcl-2inhibitors.

Non-limiting examples of therapeutic agents for Restenosis with whichbinding proteins of the invention can be combined include the following:sirolimus, paclitaxel, everolimus, tacrolimus, Zotarolimus,acetaminophen.

Non-limiting examples of therapeutic agents for Sciatica with whichbinding proteins of the invention can be combined include the following:hydrocodone bitartrate/apap, rofecoxib, cyclobenzaprine hcl,methylprednisolone, naproxen, ibuprofen, oxycodone hcl/acetaminophen,celecoxib, valdecoxib, methylprednisolone acetate, prednisone, codeinephosphate/apap, tramadol hcl/acetaminophen, metaxalone, meloxicam,methocarbamol, lidocaine hydrochloride, diclofenac sodium, gabapentin,dexamethasone, carisoprodol, ketorolac tromethamine, indomethacin,acetaminophen, diazepam, nabumetone, oxycodone hcl, tizanidine hcl,diclofenac sodium/misoprostol, propoxyphene napsylate/apap,asa/oxycod/oxycodone ter, ibuprofen/hydrocodone bit, tramadol hcl,etodolac, propoxyphene hcl, amitriptyline hcl, carisoprodol/codeinephos/asa, morphine sulfate, multivitamins, naproxen sodium, orphenadrinecitrate, and temazepam.

Examples of therapeutic agents for SLE (Lupus) in which binding proteinsof the invention can be combined include the following: NSAIDS, forexample, diclofenac, naproxen, ibuprofen, piroxicam, indomethacin; COX2inhibitors, for example, Celecoxib, rofecoxib, valdecoxib;anti-malarials, for example, hydroxychloroquine; Steroids, for example,prednisone, prednisolone, budenoside, dexamethasone; Cytotoxics, forexample, azathioprine, cyclophosphamide, mycophenolate mofetil,methotrexate; inhibitors of PDE4 or purine synthesis inhibitor, forexample Cellcept. Binding proteins of the invention, may also becombined with agents such as sulfasalazine, 5-aminosalicylic acid,olsalazine, Imuran and agents which interfere with synthesis, productionor action of proinflammatory cytokines such as IL-1, for example,caspase inhibitors like IL-1β converting enzyme inhibitors and IL-1ra.Binding proteins of the invention may also be used with T cell signalinginhibitors, for example, tyrosine kinase inhibitors; or molecules thattarget T cell activation molecules, for example, CTLA-4-IgG or anti-B7family antibodies, anti-PD-1 family antibodies. Binding proteins of theinvention, can be combined with IL-11 or anti-cytokine antibodies, forexample, fonotolizumab (anti-IFNg antibody), or anti-receptor receptorantibodies, for example, anti-IL-6 receptor antibody and antibodies toB-cell surface molecules. Antibodies of the invention or antigen bindingportion thereof may also be used with LJP 394 (abetimus), agents thatdeplete or inactivate B-cells, for example, Rituximab (anti-CD20antibody), lymphostat-B (anti-BlyS antibody), TNF antagonists, forexample, anti-TNF antibodies, Adalimumab (PCT Publication No. WO97/29131; HUMIRA), CA2 (REMICADE), CDP 571, TNFR-Ig constructs,(p75TNFRIgG (ENBREL) and p55TNFRIgG (LENERCEPT)) and bcl-2 inhibitors,because bcl-2 overexpression in transgenic mice has been demonstrated tocause a lupus like phenotype (see Marquina et al. (2004) J. Immunol.172(11):7177-7185), therefore inhibition is expected to have therapeuticeffects.

The pharmaceutical compositions of the invention may include a“therapeutically effective amount” or a “prophylactically effectiveamount” of a binding protein of the invention. A “therapeuticallyeffective amount” refers to an amount effective, at dosages and forperiods of time necessary, to achieve the desired therapeutic result. Atherapeutically effective amount of the binding protein may bedetermined by a person skilled in the art and may vary according tofactors such as the disease state, age, sex, and weight of theindividual, and the ability of the binding protein to elicit a desiredresponse in the individual. A therapeutically effective amount is alsoone in which any toxic or detrimental effects of the antibody, orantibody portion, are outweighed by the therapeutically beneficialeffects. A “prophylactically effective amount” refers to an amounteffective, at dosages and for periods of time necessary, to achieve thedesired prophylactic result. Typically, since a prophylactic dose isused in subjects prior to or at an earlier stage of disease, theprophylactically effective amount will be less than the therapeuticallyeffective amount.

Dosage regimens may be adjusted to provide the optimum desired response(e.g., a therapeutic or prophylactic response). For example, a singlebolus may be administered, several divided doses may be administeredover time or the dose may be proportionally reduced or increased asindicated by the exigencies of the therapeutic situation. It isespecially advantageous to formulate parenteral compositions in dosageunit form for ease of administration and uniformity of dosage. The term“dosage unit form” refers to physically discrete units suited as unitarydosages for the mammalian subjects to be treated; each unit containing apredetermined quantity of active compound calculated to produce thedesired therapeutic effect in association with the requiredpharmaceutical carrier. The specification for the dosage unit forms ofthe invention are dictated by and directly dependent on (a) the uniquecharacteristics of the active compound and the particular therapeutic orprophylactic effect to be achieved, and (b) the limitations inherent inthe art of compounding such an active compound for the treatment ofsensitivity in individuals.

An exemplary, non-limiting range for a therapeutically orprophylactically effective amount of a binding protein of the inventionis 0.1-20 mg/kg, for example, 1-10 mg/kg. It is to be noted that dosagevalues may vary with the type and severity of the condition to bealleviated. It is to be further understood that for any particularsubject, specific dosage regimens should be adjusted over time accordingto the individual need and the professional judgment of the personadministering or supervising the administration of the compositions, andthat dosage ranges set forth herein are exemplary only and are notintended to limit the scope or practice of the claimed composition.

It will be readily apparent to those skilled in the art that othersuitable modifications and adaptations of the methods of the inventiondescribed herein are obvious and may be made using suitable equivalentswithout departing from the scope of the invention or the embodimentsdisclosed herein. Having now described the present invention in detail,the same will be more clearly understood by reference to the followingexamples, which are included for purposes of illustration only and arenot intended to be limiting of the invention.

V. Diagnostics

The disclosure herein also provides diagnostic applications. This isfurther elucidated below.

I. Method of Assay

The present disclosure also provides a method for determining thepresence, amount or concentration of an analyte (or a fragment thereof)in a test sample using at least one DVD-Ig as described herein. Anysuitable assay as is known in the art can be used in the method.Examples include, but are not limited to, immunoassay, such as sandwichimmunoassay (e.g., monoclonal, polyclonal and/or DVD-Ig sandwichimmunoassays or any variation thereof (e.g., monoclonal/DVD-Ig,DVD-Ig/polyclonal, etc.), including radioisotope detection(radioimmunoassay (RIA)) and enzyme detection (enzyme immunoassay (EIA)or enzyme-linked immunosorbent assay (ELISA) (e.g., Quantikine ELISAassays, R&D Systems, Minneapolis, Minn.))), competitive inhibitionimmunoassay (e.g., forward and reverse), fluorescence polarizationimmunoassay (FPIA), enzyme multiplied immunoassay technique (EMIT),bioluminescence resonance energy transfer (BRET), and homogeneouschemiluminescent assay, etc. In a SELDI-based immunoassay, a capturereagent that specifically binds an analyte (or a fragment thereof) ofinterest is attached to the surface of a mass spectrometry probe, suchas a pre-activated protein chip array. The analyte (or a fragmentthereof) is then specifically captured on the biochip, and the capturedanalyte (or a fragment thereof) is detected by mass spectrometry.Alternatively, the analyte (or a fragment thereof) can be eluted fromthe capture reagent and detected by traditional MALDI (matrix-assistedlaser desorption/ionization) or by SELDI. A chemiluminescentmicroparticle immunoassay, in particular one employing the ARCHITECT®automated analyzer (Abbott Laboratories, Abbott Park, Ill.), is anexample of a preferred immunoassay.

Methods well-known in the art for collecting, handling and processingurine, blood, serum and plasma, and other body fluids, are used in thepractice of the present disclosure, for instance, when a DVD-Ig asdescribed herein is employed as an immunodiagnostic reagent and/or in ananalyte immunoassay kit. The test sample can comprise further moietiesin addition to the analyte of interest, such as antibodies, antigens,haptens, hormones, drugs, enzymes, receptors, proteins, peptides,polypeptides, oligonucleotides and/or polynucleotides. For example, thesample can be a whole blood sample obtained from a subject. It can benecessary or desired that a test sample, particularly whole blood, betreated prior to immunoassay as described herein, e.g., with apretreatment reagent. Even in cases where pretreatment is not necessary(e.g., most urine samples), pretreatment optionally can be done (e.g.,as part of a regimen on a commercial platform).

The pretreatment reagent can be any reagent appropriate for use with theimmunoassay and kits of the invention. The pretreatment optionallycomprises: (a) one or more solvents (e.g., methanol and ethylene glycol)and optionally, salt, (b) one or more solvents and salt, and optionally,detergent, (c) detergent, or (d) detergent and salt. Pretreatmentreagents are known in the art, and such pretreatment can be employed,e.g., as used for assays on Abbott TDx, AxSYM®, and ARCHITECT® analyzers(Abbott Laboratories, Abbott Park, Ill.), as described in the literature(Yatscoff et al. (1990) Clin. Chem. 36:1969-1973, and Wallemacq et al.(1999) Clin. Chem. 45:432-435), and/or as commercially available.Additionally, pretreatment can be done as described in U.S. Pat. No.5,135,875; EU Patent Pubublication No. EU0471293; U.S. Pat. No.6,660,843; and US Patent Application No. 20080020401. The pretreatmentreagent can be a heterogeneous agent or a homogeneous agent.

With use of a heterogeneous pretreatment reagent, the pretreatmentreagent precipitates analyte binding protein (e.g., protein that canbind to an analyte or a fragment thereof) present in the sample. Such apretreatment step comprises removing any analyte binding protein byseparating from the precipitated analyte binding protein the supernatantof the mixture formed by addition of the pretreatment agent to sample.In such an assay, the supernatant of the mixture absent any bindingprotein is used in the assay, proceeding directly to the antibodycapture step.

With use of a homogeneous pretreatment reagent there is no suchseparation step. The entire mixture of test sample and pretreatmentreagent are contacted with a labeled specific binding partner foranalyte (or a fragment thereof), such as a labeled anti-analyte antibody(or an antigenically reactive fragment thereof). The pretreatmentreagent employed for such an assay typically is diluted in thepretreated test sample mixture, either before or during capture by thefirst specific binding partner. Despite such dilution, a certain amountof the pretreatment reagent is still present (or remains) in the testsample mixture during capture. According to the invention, the labeledspecific binding partner can be a DVD-Ig (or a fragment, a variant, or afragment of a variant thereof).

In a heterogeneous format, after the test sample is obtained from asubject, a first mixture is prepared. The mixture contains the testsample being assessed for an analyte (or a fragment thereof) and a firstspecific binding partner, wherein the first specific binding partner andany analyte contained in the test sample form a first specific bindingpartner-analyte complex. Preferably, the first specific binding partneris an anti-analyte antibody or a fragment thereof. The first specificbinding partner can be a DVD-Ig (or a fragment, a variant, or a fragmentof a variant thereof) as described herein. The order in which the testsample and the first specific binding partner are added to form themixture is not critical. Preferably, the first specific binding partneris immobilized on a solid phase. The solid phase used in the immunoassay(for the first specific binding partner and, optionally, the secondspecific binding partner) can be any solid phase known in the art, suchas, but not limited to, a magnetic particle, a bead, a test tube, amicrotiter plate, a cuvette, a membrane, a scaffolding molecule, a film,a filter paper, a disc and a chip.

After the mixture containing the first specific binding partner-analytecomplex is formed, any unbound analyte is removed from the complex usingany technique known in the art. For example, the unbound analyte can beremoved by washing. Desirably, however, the first specific bindingpartner is present in excess of any analyte present in the test sample,such that all analyte that is present in the test sample is bound by thefirst specific binding partner.

After any unbound analyte is removed, a second specific binding partneris added to the mixture to form a first specific bindingpartner-analyte-second specific binding partner complex. The secondspecific binding partner is preferably an anti-analyte antibody thatbinds to an epitope on analyte that differs from the epitope on analytebound by the first specific binding partner. Moreover, also preferably,the second specific binding partner is labeled with or contains adetectable label as described above. The second specific binding partnercan be a DVD-Ig (or a fragment, a variant, or a fragment of a variantthereof) as described herein.

Any suitable detectable label as is known in the art can be used. Forexample, the detectable label can be a radioactive label (such as 3H,125I, 35S, 14C, 32P, and 33P), an enzymatic label (such as horseradishperoxidase, alkaline peroxidase, glucose 6-phosphate dehydrogenase, andthe like), a chemiluminescent label (such as acridinium esters,thioesters, or sulfonamides; luminol, isoluminol, phenanthridiniumesters, and the like), a fluorescent label (such as fluorescein (e.g.,5-fluorescein, 6-carboxyfluorescein, 3′6-carboxyfluorescein,5(6)-carboxyfluorescein, 6-hexachloro-fluorescein,6-tetrachlorofluorescein, fluorescein isothiocyanate, and the like)),rhodamine, phycobiliproteins, R-phycoerythrin, quantum dots (e.g., zincsulfide-capped cadmium selenide), a thermometric label, or animmuno-polymerase chain reaction label. An introduction to labels,labeling procedures and detection of labels is found in Polak and VanNoorden, Introduction to Immunocytochemistry, 2nd ed., Springer Verlag,N.Y. (1997), and in Haugland, Handbook of Fluorescent Probes andResearch Chemicals (1996), which is a combined handbook and cataloguepublished by Molecular Probes, Inc., Eugene, Oreg. A fluorescent labelcan be used in FPIA (U.S. Pat. Nos. 5,593,896; 5,573,904; 5,496,925;5,359,093; and 5,352,803). An acridinium compound can be used as adetectable label in a homogeneous or heterogeneous chemiluminescentassay (Adamczyk et al. (2006) Bioorg. Med. Chem. Lett. 16:1324-1328;Adamczyk et al. (2004) Bioorg. Med. Chem. Lett. 4:2313-2317; Adamczyk etal. (2004) Biorg. Med. Chem. Lett. 14: 3917-3921; and Adamczyk et al.(2003) Org. Lett. 5:3779-3782).

A preferred acridinium compound is an acridinium-9-carboxamide. Methodsfor preparing acridinium 9-carboxamides are described in Mattingly(1991) J. Biolumin. Chemilumin. 6:107-114; Adamczyk et al. (1998) J.Org. Chem. 63:5636-5639; Adamczyk et al. (1999) Tetrahedron55:10899-10914; Adamczyk et al. (1999) Org. Lett. 1:779-781; Adamczyk etal. (2000) Bioconjug. Chem. 11:714-724; Mattingly et al., InLuminescence Biotechnology: Instruments and Applications; Dyke, Ed.(2002) CRC Press: Boca Raton, pp. 77-105; Adamczyk et al. (2003) Org.Lett. 5: 3779-3782; and U.S. Pat. Nos. 5,468,646; 5,543,524; and5,783,699. Another preferred acridinium compound is anacridinium-9-carboxylate aryl ester. An example of anacridinium-9-carboxylate aryl ester is10-methyl-9-(phenoxycarbonyl)acridinium fluorosulfonate (available fromCayman Chemical, Ann Arbor, Mich.). Methods for preparing acridinium9-carboxylate aryl esters are described in McCapra et al. (1965)Photochem. Photobiol. 4:1111-21; Razavi et al. (2000) Luminescence15:245-249; Razavi et al. (2000) Luminescence 15:239-244; and U.S. Pat.No. 5,241,070. Further details regarding acridinium-9-carboxylate arylester and its use are set forth in US Patent Publication No.20080248493.

Chemiluminescent assays (e.g., using acridinium as described above orother chemiluminescent agents) can be performed in accordance with themethods described in Adamczyk et al. (2006) Anal. Chim. Acta579(1):61-67. While any suitable assay format can be used, a microplatechemiluminometer (Mithras LB-940, Berthold Technologies USA, LLC, OakRidge, Tenn.) enables the assay of multiple samples of small volumesrapidly.

The order in which the test sample and the specific binding partner(s)are added to form the mixture for chemiluminescent assay is notcritical. If the first specific binding partner is detectably labeledwith a chemiluminescent agent such as an acridinium compound, detectablylabeled first specific binding partner-analyte complexes form.Alternatively, if a second specific binding partner is used and thesecond specific binding partner is detectably labeled with achemiluminescent agent such as an acridinium compound, detectablylabeled first specific binding partner-analyte-second specific bindingpartner complexes form. Any unbound specific binding partner, whetherlabeled or unlabeled, can be removed from the mixture using anytechnique known in the art, such as washing.

Hydrogen peroxide can be generated in situ in the mixture or provided orsupplied to the mixture (e.g., the source of the hydrogen peroxide beingone or more buffers or other solutions that are known to containhydrogen peroxide) before, simultaneously with, or after the addition ofan above-described acridinium compound. Hydrogen peroxide can begenerated in situ in a number of ways such as would be apparent to oneskilled in the art.

Upon the simultaneous or subsequent addition of at least one basicsolution to the sample, a detectable signal, namely, a chemiluminescentsignal, indicative of the presence of analyte is generated. The basicsolution contains at least one base and has a pH greater than or equalto 10, preferably, greater than or equal to 12. Examples of basicsolutions include, but are not limited to, sodium hydroxide, potassiumhydroxide, calcium hydroxide, ammonium hydroxide, magnesium hydroxide,sodium carbonate, sodium bicarbonate, calcium hydroxide, calciumcarbonate, and calcium bicarbonate. The amount of basic solution addedto the sample depends on the concentration of the basic solution. Basedon the concentration of the basic solution used, one skilled in the artcan easily determine the amount of basic solution to add to the sample.

The chemiluminescent signal that is generated can be detected usingroutine techniques known to those skilled in the art. Based on theintensity of the signal generated, the amount of analyte in the samplecan be quantified. Specifically, the amount of analyte in the sample isproportional to the intensity of the signal generated. The amount ofanalyte present can be quantified by comparing the amount of lightgenerated to a standard curve for analyte or by comparison to areference standard. The standard curve can be generated using serialdilutions or solutions of known concentrations of analyte by massspectroscopy, gravimetric methods, and other techniques known in theart. While the above is described with emphasis on use of an acridiniumcompound as the chemiluminescent agent, one of ordinary skill in the artcan readily adapt this description for use of other chemiluminescentagents.

Analyte immunoassays generally can be conducted using any format knownin the art, such as, but not limited to, a sandwich format.Specifically, in one immunoassay format, at least two antibodies areemployed to separate and quantify analyte, such as human analyte, or afragment thereof in a sample. More specifically, the at least twoantibodies bind to different epitopes on an analyte (or a fragmentthereof) forming an immune complex, which is referred to as a“sandwich.” Generally, in the immunoassays one or more antibodies can beused to capture the analyte (or a fragment thereof) in the test sample(these antibodies are frequently referred to as a “capture” antibody or“capture” antibodies) and one or more antibodies can be used to bind adetectable (namely, quantifiable) label to the sandwich (theseantibodies are frequently referred to as the “detection antibody,” the“detection antibodies,” the “conjugate,” or the “conjugates”). Thus, inthe context of a sandwich immunoassay format, a binding protein orDVD-Ig (or a fragment, a variant, or a fragment of a variant thereof) asdescribed herein can be used as a capture antibody, a detectionantibody, or both. For example, one binding protein or DVD-Ig having adomain that can bind a first epitope on an analyte (or a fragmentthereof) can be used as a capture antibody and/or another bindingprotein or DVD-Ig having a domain that can bind a second epitope on ananalyte (or a fragment thereof) can be used as a detection antibody. Inthis regard, a binding protein or DVD-Ig having a first domain that canbind a first epitope on an analyte (or a fragment thereof) and a seconddomain that can bind a second epitope on an analyte (or a fragmentthereof) can be used as a capture antibody and/or a detection antibody.Alternatively, one binding protein or DVD-Ig having a first domain thatcan bind an epitope on a first analyte (or a fragment thereof) and asecond domain that can bind an epitope on a second analyte (or afragment thereof) can be used as a capture antibody and/or a detectionantibody to detect, and optionally quantify, two or more analytes. Inthe event that an analyte can be present in a sample in more than oneform, such as a monomeric form and a dimeric/multimeric form, which canbe homomeric or heteromeric, one binding protein or DVD-Ig having adomain that can bind an epitope that is only exposed on the monomericform and another binding protein or DVD-Ig having a domain that can bindan epitope on a different part of a dimeric/multimeric form can be usedas capture antibodies and/or detection antibodies, thereby enabling thedetection, and optional quantification, of different forms of a givenanalyte. Furthermore, employing DVD-Igs with differential affinitieswithin a single binding protein or DVD-Ig and/or between bindingproteins or DVD-Igs can provide an avidity advantage. In the context ofimmunoassays as described herein, it generally may be helpful or desiredto incorporate one or more linkers within the structure of a bindingprotein or DVD-Ig. When present, optimally the linker should be ofsufficient length and structural flexibility to enable binding of anepitope by the inner domains as well as binding of another epitope bythe outer domains. In this regard, if a binding protein or DVD-Ig canbind two different analytes and one analyte is larger than the other,desirably the larger analyte is bound by the outer domains.

Generally speaking, a sample being tested for (for example, suspected ofcontaining) analyte (or a fragment thereof) can be contacted with atleast one capture antibody (or antibodies) and at least one detectionantibody (which can be a second detection antibody or a third detectionantibody or even a successively numbered antibody, e.g., as where thecapture and/or detection antibody comprise multiple antibodies) eithersimultaneously or sequentially and in any order. For example, the testsample can be first contacted with at least one capture antibody andthen (sequentially) with at least one detection antibody. Alternatively,the test sample can be first contacted with at least one detectionantibody and then (sequentially) with at least one capture antibody. Inyet another alternative, the test sample can be contacted simultaneouslywith a capture antibody and a detection antibody.

In the sandwich assay format, a sample suspected of containing analyte(or a fragment thereof) is first brought into contact with at least onefirst capture antibody under conditions that allow the formation of afirst antibody/analyte complex. If more than one capture antibody isused, a first capture antibody/analyte complex comprising two or morecapture antibodies is formed. In a sandwich assay, the antibodies, i.e.,preferably, the at least one capture antibody, are used in molar excessamounts of the maximum amount of analyte (or a fragment thereof)expected in the test sample. For example, from about 5 μg to about 1 mgof antibody per mL of buffer (e.g., microparticle coating buffer) can beused.

Competitive inhibition immunoassays, which are often used to measuresmall analytes because binding by only one antibody is required,comprise sequential and classic formats. In a sequential competitiveinhibition immunoassay a capture antibody to an analyte of interest iscoated onto a well of a microtiter plate or other solid support. Whenthe sample containing the analyte of interest is added to the well, theanalyte of interest binds to the capture antibody. After washing, aknown amount of labeled (e.g., biotin or horseradish peroxidase (HRP))analyte is added to the well. A substrate for an enzymatic label isnecessary to generate a signal. An example of a suitable substrate forHRP is 3,3′,5,5′-tetramethylbenzidine (TMB). After washing, the signalgenerated by the labeled analyte is measured and is inverselyproportional to the amount of analyte in the sample. In a classiccompetitive inhibition immunoassay an antibody to an analyte of interestis coated onto a solid support (e.g., a well of a microtiter plate).However, unlike the sequential competitive inhibition immunoassay, thesample and the labeled analyte are added to the well at the same time.Any analyte in the sample competes with labeled analyte for binding tothe capture antibody. After washing, the signal generated by the labeledanalyte is measured and is inversely proportional to the amount ofanalyte in the sample.

Optionally, prior to contacting the test sample with the at least onecapture antibody (for example, the first capture antibody), the at leastone capture antibody can be bound to a solid support, which facilitatesthe separation of the first antibody/analyte (or a fragment thereof)complex from the test sample. The substrate to which the captureantibody is bound can be any suitable solid support or solid phase thatfacilitates separation of the capture antibody-analyte complex from thesample.

Examples include a well of a plate, such as a microtiter plate, a testtube, a porous gel (e.g., silica gel, agarose, dextran, or gelatin), apolymeric film (e.g., polyacrylamide), beads (e.g., polystyrene beads ormagnetic beads), a strip of a filter/membrane (e.g., nitrocellulose ornylon), microparticles (e.g., latex particles, magnetizablemicroparticles (e.g., microparticles having ferric oxide or chromiumoxide cores and homo- or hetero-polymeric coats and radii of about 1-10microns). The substrate can comprise a suitable porous material with asuitable surface affinity to bind antigens and sufficient porosity toallow access by detection antibodies. A microporous material isgenerally preferred, although a gelatinous material in a hydrated statecan be used. Such porous substrates are preferably in the form of sheetshaving a thickness of about 0.01 to about 0.5 mm, preferably about 0.1mm. While the pore size may vary quite a bit, preferably the pore sizeis from about 0.025 to about 15 microns, more preferably from about 0.15to about 15 microns. The surface of such substrates can be activated bychemical processes that cause covalent linkage of an antibody to thesubstrate. Irreversible binding, generally by adsorption throughhydrophobic forces, of the antigen or the antibody to the substrateresults; alternatively, a chemical coupling agent or other means can beused to bind covalently the antibody to the substrate, provided thatsuch binding does not interfere with the ability of the antibody to bindto analyte. Alternatively, the antibody can be bound withmicroparticles, which have been previously coated with streptavidin(e.g., DYNAL® Magnetic Beads, Invitrogen, Carlsbad, Calif.) or biotin(e.g., using Power-Bind™-SA-MP streptavidin-coated microparticles(Seradyn, Indianapolis, Ind.)) or anti-species-specific monoclonalantibodies. If necessary, the substrate can be derivatized to allowreactivity with various functional groups on the antibody. Suchderivatization requires the use of certain coupling agents, examples ofwhich include, but are not limited to, maleic anhydride,N-hydroxysuccinimide, and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide. If desired, one or more capture reagents, such asantibodies (or fragments thereof), each of which is specific foranalyte(s) can be attached to solid phases in different physical oraddressable locations (e.g., such as in a biochip configuration (see,e.g., U.S. Pat. Nos. 6,225,047; 6,329,209; and 5,242,828; and PCTPublication No. WO 99/51773 and WO 00/56934). If the capture reagent isattached to a mass spectrometry probe as the solid support, the amountof analyte bound to the probe can be detected by laser desorptionionization mass spectrometry. Alternatively, a single column can bepacked with different beads, which are derivatized with the one or morecapture reagents, thereby capturing the analyte in a single place (see,antibody-derivatized, bead-based technologies, e.g., the xMAP technologyof Luminex (Austin, Tex.)).

After the test sample being assayed for analyte (or a fragment thereof)is brought into contact with the at least one capture antibody (forexample, the first capture antibody), the mixture is incubated in orderto allow for the formation of a first antibody (or multipleantibody)-analyte (or a fragment thereof) complex. The incubation can becarried out at a pH of from about 4.5 to about 10.0, at a temperature offrom about 2° C. to about 45° C., and for a period from at least aboutone (1) minute to about eighteen (18) hours, preferably from about 1 toabout 24 minutes, most preferably for about 4 to about 18 minutes. Theimmunoassay described herein can be conducted in one step (meaning thetest sample, at least one capture antibody and at least one detectionantibody are all added sequentially or simultaneously to a reactionvessel) or in more than one step, such as two steps, three steps, etc.

After formation of the (first or multiple) capture antibody/analyte (ora fragment thereof) complex, the complex is then contacted with at leastone detection antibody under conditions which allow for the formation ofa (first or multiple) capture antibody/analyte (or a fragmentthereof)/second detection antibody complex). While captioned for clarityas the “second” antibody (e.g., second detection antibody), in fact,where multiple antibodies are used for capture and/or detection, the atleast one detection antibody can be the second, third, fourth, etc.antibodies used in the immunoassay. If the capture antibody/analyte (ora fragment thereof) complex is contacted with more than one detectionantibody, then a (first or multiple) capture antibody/analyte (or afragment thereof)/(multiple) detection antibody complex is formed. Aswith the capture antibody (e.g., the first capture antibody), when theat least one (e.g., second and any subsequent) detection antibody isbrought into contact with the capture antibody/analyte (or a fragmentthereof) complex, a period of incubation under conditions similar tothose described above is required for the formation of the (first ormultiple) capture antibody/analyte (or a fragment thereof)/(second ormultiple) detection antibody complex. Preferably, at least one detectionantibody contains a detectable label. The detectable label can be boundto the at least one detection antibody (e.g., the second detectionantibody) prior to, simultaneously with, or after the formation of the(first or multiple) capture antibody/analyte (or a fragmentthereof)/(second or multiple) detection antibody complex. Any detectablelabel known in the art can be used (see discussion above, including ofthe Polak and Van Noorden (1997) and Haugland (1996) references).

The detectable label can be bound to the antibodies either directly orthrough a coupling agent. An example of a coupling agent that can beused is EDAC (1-ethyl-3-(3-dimethylaminopropyl) carbodiimide,hydrochloride), which is commercially available from Sigma-Aldrich, St.Louis, Mo. Other coupling agents that can be used are known in the art.Methods for binding a detectable label to an antibody are known in theart. Additionally, many detectable labels can be purchased orsynthesized that already contain end groups that facilitate the couplingof the detectable label to the antibody, such as CPSP-Acridinium Ester(i.e., 9-[N-tosyl-N-(3-carboxypropyl)]-10-(3-sulfopropyl)acridiniumcarboxamide) or SPSP-Acridinium Ester (i.e.,N10-(3-sulfopropyl)-N-(3-sulfopropyl)-acridinium-9-carboxamide).

The (first or multiple) capture antibody/analyte/(second or multiple)detection antibody complex can be, but does not have to be, separatedfrom the remainder of the test sample prior to quantification of thelabel. For example, if the at least one capture antibody (e.g., thefirst capture antibody) is bound to a solid support, such as a well or abead, separation can be accomplished by removing the fluid (of the testsample) from contact with the solid support. Alternatively, if the atleast first capture antibody is bound to a solid support, it can besimultaneously contacted with the analyte-containing sample and the atleast one second detection antibody to form a first (multiple)antibody/analyte/second (multiple) antibody complex, followed by removalof the fluid (test sample) from contact with the solid support. If theat least one first capture antibody is not bound to a solid support,then the (first or multiple) capture antibody/analyte/(second ormultiple) detection antibody complex does not have to be removed fromthe test sample for quantification of the amount of the label.

After formation of the labeled capture antibody/analyte/detectionantibody complex (e.g., the first capture antibody/analyte/seconddetection antibody complex), the amount of label in the complex isquantified using techniques known in the art. For example, if anenzymatic label is used, the labeled complex is reacted with a substratefor the label that gives a quantifiable reaction such as the developmentof color. If the label is a radioactive label, the label is quantifiedusing appropriate means, such as a scintillation counter. If the labelis a fluorescent label, the label is quantified by stimulating the labelwith a light of one color (which is known as the “excitationwavelength”) and detecting another color (which is known as the“emission wavelength”) that is emitted by the label in response to thestimulation. If the label is a chemiluminescent label, the label isquantified by detecting the light emitted either visually or by usingluminometers, x-ray film, high speed photographic film, a CCD camera,etc. Once the amount of the label in the complex has been quantified,the concentration of analyte or a fragment thereof in the test sample isdetermined by appropriate means, such as by use of a standard curve thathas been generated using serial dilutions of analyte or a fragmentthereof of known concentration. Other than using serial dilutions ofanalyte or a fragment thereof, the standard curve can be generatedgravimetrically, by mass spectroscopy and by other techniques known inthe art.

In a chemiluminescent microparticle assay employing the ARCHITECT®analyzer, the conjugate diluent pH should be about 6.0+/−0.2, themicroparticle coating buffer should be maintained at about roomtemperature (i.e., at from about 17 to about 27° C.), the microparticlecoating buffer pH should be about 6.5+/−0.2, and the microparticlediluent pH should be about 7.8+/−0.2. Solids preferably are less thanabout 0.2%, such as less than about 0.15%, less than about 0.14%, lessthan about 0.13%, less than about 0.12%, or less than about 0.11%, suchas about 0.10%.

FPIAs are based on competitive binding immunoassay principles. Afluorescently labeled compound, when excited by a linearly polarizedlight, will emit fluorescence having a degree of polarization inverselyproportional to its rate of rotation. When a fluorescently labeledtracer-antibody complex is excited by a linearly polarized light, theemitted light remains highly polarized because the fluorophore isconstrained from rotating between the time light is absorbed and thetime light is emitted. When a “free” tracer compound (i.e., a compoundthat is not bound to an antibody) is excited by linearly polarizedlight, its rotation is much faster than the correspondingtracer-antibody conjugate (or tracer binding protein and/or tracerDVD-Ig) produced in a competitive binding immunoassay. FPIAs areadvantageous over RIAs inasmuch as there are no radioactive substancesrequiring special handling and disposal. In addition, FPIAs arehomogeneous assays that can be easily and rapidly performed.

In view of the above, a method of determining the presence, amount, orconcentration of analyte (or a fragment thereof) in a test sample isprovided. The method comprises assaying the test sample for an analyte(or a fragment thereof) by an assay (i) employing (i′) at least one ofan antibody, a fragment of an antibody that can bind to an analyte, avariant of an antibody that can bind to an analyte, a fragment of avariant of an antibody that can bind to an analyte (or tracer bindingprotein and/or tracer DVD-Ig), and a DVD-Ig (or a fragment, a variant,or a fragment of a variant thereof) that can bind to an analyte, and(ii′) at least one detectable label and (ii) comprising comparing asignal generated by the detectable label as a direct or indirectindication of the presence, amount or concentration of analyte (or afragment thereof) in the test sample to a signal generated as a director indirect indication of the presence, amount or concentration ofanalyte (or a fragment thereof) in a control or calibrator. Thecalibrator is optionally part of a series of calibrators, in which eachof the calibrators differs from the other calibrators by theconcentration of analyte.

The method can comprise (i) contacting the test sample with at least onefirst specific binding partner for analyte (or a fragment thereof)selected from the group consisting of an antibody, a fragment of anantibody that can bind to an analyte, a variant of an antibody that canbind to an analyte, a fragment of a variant of an antibody that can bindto an analyte, and a DVD-Ig (or a fragment, a variant, or a fragment ofa variant thereof) that can bind to an analyte so as to form a firstspecific binding partner/analyte (or fragment thereof) complex, (ii)contacting the first specific binding partner/analyte (or fragmentthereof) complex with at least one second specific binding partner foranalyte (or fragment thereof) selected from the group consisting of adetectably labeled anti-analyte antibody, a detectably labeled fragmentof an anti-analyte antibody that can bind to analyte, a detectablylabeled variant of an anti-analyte antibody that can bind to analyte, adetectably labeled fragment of a variant of an anti-analyte antibodythat can bind to analyte, and a detectably labeled DVD-Ig (or afragment, a variant, or a fragment of a variant thereof) so as to form afirst specific binding partner/analyte (or fragment thereof)/secondspecific binding partner complex, and (iii) determining the presence,amount or concentration of analyte in the test sample by detecting ormeasuring the signal generated by the detectable label in the firstspecific binding partner/analyte (or fragment thereof)/second specificbinding partner complex formed in (ii). A method in which at least onefirst specific binding partner for analyte (or a fragment thereof)and/or at least one second specific binding partner for analyte (or afragment thereof) is a DVD-Ig (or a fragment, a variant, or a fragmentof a variant thereof) as described herein can be preferred.

Alternatively, the method can comprise contacting the test sample withat least one first specific binding partner for analyte (or a fragmentthereof) selected from the group consisting of an antibody, a fragmentof an antibody that can bind to an analyte, a variant of an antibodythat can bind to an analyte, a fragment of a variant of an antibody thatcan bind to an analyte, and a DVD-Ig (or a fragment, a variant, or afragment of a variant thereof) and simultaneously or sequentially, ineither order, contacting the test sample with at least one secondspecific binding partner, which can compete with analyte (or a fragmentthereof) for binding to the at least one first specific binding partnerand which is selected from the group consisting of a detectably labeledanalyte, a detectably labeled fragment of analyte that can bind to thefirst specific binding partner, a detectably labeled variant of analytethat can bind to the first specific binding partner, and a detectablylabeled fragment of a variant of analyte that can bind to the firstspecific binding partner. Any analyte (or a fragment thereof) present inthe test sample and the at least one second specific binding partnercompete with each other to form a first specific binding partner/analyte(or fragment thereof) complex and a first specific bindingpartner/second specific binding partner complex, respectively. Themethod further comprises determining the presence, amount orconcentration of analyte in the test sample by detecting or measuringthe signal generated by the detectable label in the first specificbinding partner/second specific binding partner complex formed in (ii),wherein the signal generated by the detectable label in the firstspecific binding partner/second specific binding partner complex isinversely proportional to the amount or concentration of analyte in thetest sample.

The above methods can further comprise diagnosing, prognosticating, orassessing the efficacy of a therapeutic/prophylactic treatment of apatient from whom the test sample was obtained. If the method furthercomprises assessing the efficacy of a therapeutic/prophylactic treatmentof the patient from whom the test sample was obtained, the methodoptionally further comprises modifying the therapeutic/prophylactictreatment of the patient as needed to improve efficacy. The method canbe adapted for use in an automated system or a semi-automated system.

More specifically, a method of determining the presence, amount orconcentration of an antigen (or a fragment thereof) in a test sample isprovided. The method comprises assaying the test sample for the antigen(or a fragment thereof) by an immunoassay. The immunoassay (i) employsat least one binding protein and at least one detectable label and (ii)comprises comparing a signal generated by the detectable label as adirect or indirect indication of the presence, amount or concentrationof the antigen (or a fragment thereof) in the test sample to a signalgenerated as a direct or indirect indication of the presence, amount orconcentration of the antigen (or a fragment thereof) in a control or acalibrator. The calibrator is optionally part of a series of calibratorsin which each of the calibrators differs from the other calibrators inthe series by the concentration of the antigen (or a fragment thereof).One of the at least one binding protein (i′) comprises a polypeptidechain comprising VD1-(X1)n-VD2-C-(X2)n, in which VD1 is a first heavychain variable domain obtained from a first parent antibody (or antigenbinding portion thereof), VD2 is a second heavy chain variable domainobtained from a second parent antibody (or antigen binding portionthereof), which can be the same as or different from the first parentantibody, C is a heavy chain constant domain, (X1)n is a linker, whichis optionally present and, when present, is other than CH1, and (X2)n isan Fc region, which is optionally present, and (ii′) can bind a pair ofantigens. The method can comprise (i) contacting the test sample with atleast one capture agent, which binds to an epitope on the antigen (or afragment thereof) so as to form a capture agent/antigen (or a fragmentthereof) complex, (ii) contacting the capture agent/antigen (or afragment thereof) complex with at least one detection agent, whichcomprises a detectable label and binds to an epitope on the antigen (ora fragment thereof) that is not bound by the capture agent, to form acapture agent/antigen (or a fragment thereof)/detection agent complex,and (iii) determining the presence, amount or concentration of theantigen (or a fragment thereof) in the test sample based on the signalgenerated by the detectable label in the capture agent/antigen (or afragment thereof)/detection agent complex formed in (ii), wherein atleast one capture agent and/or at least one detection agent is the atleast one binding protein. Alternatively, the method can comprise (i)contacting the test sample with at least one capture agent, which bindsto an epitope on the antigen (or a fragment thereof) so as to form acapture agent/antigen (or a fragment thereof) complex, andsimultaneously or sequentially, in either order, contacting the testsample with detectably labeled antigen (or a fragment thereof), whichcan compete with any antigen (or a fragment thereof) in the test samplefor binding to the at least one capture agent, wherein any antigen (or afragment thereof) present in the test sample and the detectably labeledantigen compete with each other to form a capture agent/antigen (or afragment thereof) complex and a capture agent/detectably labeled antigen(or a fragment thereof) complex, respectively, and (ii) determining thepresence, amount or concentration of the antigen (or a fragment thereof)in the test sample based on the signal generated by the detectable labelin the capture agent/detectably labeled antigen (or a fragment thereof)complex formed in (ii), wherein at least one capture agent is the atleast one binding protein and wherein the signal generated by thedetectable label in the capture agent/detectably labeled antigen (or afragment thereof) complex is inversely proportional to the amount orconcentration of antigen (or a fragment thereof) in the test sample. Thetest sample can be from a patient, in which case the method can furthercomprise diagnosing, prognosticating, or assessing the efficacy oftherapeutic/prophylactic treatment of the patient. If the method furthercomprises assessing the efficacy of therapeutic/prophylactic treatmentof the patient, the method optionally further comprises modifying thetherapeutic/prophylactic treatment of the patient as needed to improveefficacy. The method can be adapted for use in an automated system or asemi-automated system.

Another method of determining the presence, amount or concentration ofan antigen (or a fragment thereof) in a test sample is provided. Themethod comprises assaying the test sample for the antigen (or a fragmentthereof) by an immunoassay. The immunoassay (i) employs at least onebinding protein and at least one detectable label and (ii) comprisescomparing a signal generated by the detectable label as a direct orindirect indication of the presence, amount or concentration of theantigen (or a fragment thereof) in the test sample to a signal generatedas a direct or indirect indication of the presence, amount orconcentration of the antigen (or a fragment thereof) in a control or acalibrator. The calibrator is optionally part of a series of calibratorsin which each of the calibrators differs from the other calibrators inthe series by the concentration of the antigen (or a fragment thereof).One of the at least one binding protein (i′) comprises a polypeptidechain comprising VD1-(X1)n-VD2-C-(X2)n, in which VD1 is a first lightchain variable domain obtained from a first parent antibody (or antigenbinding portion thereof), VD2 is a second light chain variable domainobtained from a second parent antibody (or antigen binding portionthereof), which can be the same as or different from the first parentantibody, C is a light chain constant domain, (X1)n is a linker, whichis optionally present and, when present, is other than CH1, and (X2)n isan Fc region, which is optionally present, and (ii′) can bind a pair ofantigens. The method can comprise (i) contacting the test sample with atleast one capture agent, which binds to an epitope on the antigen (or afragment thereof) so as to form a capture agent/antigen (or a fragmentthereof) complex, (ii) contacting the capture agent/antigen (or afragment thereof) complex with at least one detection agent, whichcomprises a detectable label and binds to an epitope on the antigen (ora fragment thereof) that is not bound by the capture agent, to form acapture agent/antigen (or a fragment thereof)/detection agent complex,and (iii) determining the presence, amount or concentration of theantigen (or a fragment thereof) in the test sample based on the signalgenerated by the detectable label in the capture agent/antigen (or afragment thereof)/detection agent complex formed in (ii), wherein atleast one capture agent and/or at least one detection agent is the atleast one binding protein. Alternatively, the method can comprise (i)contacting the test sample with at least one capture agent, which bindsto an epitope on the antigen (or a fragment thereof) so as to form acapture agent/antigen (or a fragment thereof) complex, andsimultaneously or sequentially, in either order, contacting the testsample with detectably labeled antigen (or a fragment thereof), whichcan compete with any antigen (or a fragment thereof) in the test samplefor binding to the at least one capture agent, wherein any antigen (or afragment thereof) present in the test sample and the detectably labeledantigen compete with each other to form a capture agent/antigen (or afragment thereof) complex and a capture agent/detectably labeled antigen(or a fragment thereof) complex, respectively, and (ii) determining thepresence, amount or concentration of the antigen (or a fragment thereof)in the test sample based on the signal generated by the detectable labelin the capture agent/detectably labeled antigen (or a fragment thereof)complex formed in (ii), wherein at least one capture agent is the atleast one binding protein and wherein the signal generated by thedetectable label in the capture agent/detectably labeled antigen (or afragment thereof) complex is inversely proportional to the amount orconcentration of antigen (or a fragment thereof) in the test sample. Ifthe test sample is from a patient, the method can further comprisediagnosing, prognosticating, or assessing the efficacy oftherapeutic/prophylactic treatment of the patient. If the method furthercomprises assessing the efficacy of therapeutic/prophylactic treatmentof the patient, the method optionally further comprises modifying thetherapeutic/prophylactic treatment of the patient as needed to improveefficacy. The method can be adapted for use in an automated system or asemi-automated system.

Yet another method of determining the presence, amount or concentrationof an antigen (or a fragment thereof) in a test sample is provided. Themethod comprises assaying the test sample for the antigen (or a fragmentthereof) by an immunoassay. The immunoassay (i) employs at least onebinding protein and at least one detectable label and (ii) comprisescomparing a signal generated by the detectable label as a direct orindirect indication of the presence, amount or concentration of theantigen (or a fragment thereof) in the test sample to a signal generatedas a direct or indirect indication of the presence, amount orconcentration of the antigen (or a fragment thereof) in a control or acalibrator. The calibrator is optionally part of a series of calibratorsin which each of the calibrators differs from the other calibrators inthe series by the concentration of the antigen (or a fragment thereof).One of the at least one binding protein (i′) comprises a firstpolypeptide chain and a second polypeptide chain, wherein the firstpolypeptide chain comprises a first VD1-(X1)n-VD2-C-(X2)n, in which VD1is a first heavy chain variable domain obtained from a first parentantibody (or antigen binding portion thereof), VD2 is a second heavychain variable domain obtained from a second parent antibody (or antigenbinding portion thereof), which can be the same as or different from thefirst parent antibody, C is a heavy chain constant domain, (X1)n is alinker, which is optionally present and, when present, is other thanCH1, and (X2)n is an Fc region, which is optionally present, and whereinthe second polypeptide chain comprises a second VD1-(X1)n-VD2-C-(X2)n,in which VD1 is a first light chain variable domain obtained from afirst parent antibody (or antigen binding portion thereof), VD2 is asecond light chain variable domain obtained from a second parentantibody (or antigen binding portion thereof), which can be the same asor different from the first parent antibody, C is a light chain constantdomain, (X1)n is a linker, which is optionally present and, whenpresent, is other than CH1, and (X2)n is an Fc region, which isoptionally present, and (ii′) can bind a pair of antigens. The methodcan comprise (i) contacting the test sample with at least one captureagent, which binds to an epitope on the antigen (or a fragment thereof)so as to form a capture agent/antigen (or a fragment thereof) complex,(ii) contacting the capture agent/antigen (or a fragment thereof)complex with at least one detection agent, which comprises a detectablelabel and binds to an epitope on the antigen (or a fragment thereof)that is not bound by the capture agent, to form a capture agent/antigen(or a fragment thereof)/detection agent complex, and (iii) determiningthe presence, amount or concentration of the antigen (or a fragmentthereof) in the test sample based on the signal generated by thedetectable label in the capture agent/antigen (or a fragmentthereof)/detection agent complex formed in (ii), wherein at least onecapture agent and/or at least one detection agent is the at least onebinding protein. Alternatively, the method can comprise (i) contactingthe test sample with at least one capture agent, which binds to anepitope on the antigen (or a fragment thereof) so as to form a captureagent/antigen (or a fragment thereof) complex, and simultaneously orsequentially, in either order, contacting the test sample withdetectably labeled antigen (or a fragment thereof), which can competewith any antigen (or a fragment thereof) in the test sample for bindingto the at least one capture agent, wherein any antigen (or a fragmentthereof) present in the test sample and the detectably labeled antigencompete with each other to form a capture agent/antigen (or a fragmentthereof) complex and a capture agent/detectably labeled antigen (or afragment thereof) complex, respectively, and (ii) determining thepresence, amount or concentration of the antigen (or a fragment thereof)in the test sample based on the signal generated by the detectable labelin the capture agent/detectably labeled antigen (or a fragment thereof)complex formed in (ii), wherein at least one capture agent is the atleast one binding protein and wherein the signal generated by thedetectable label in the capture agent/detectably labeled antigen (or afragment thereof) complex is inversely proportional to the amount orconcentration of antigen (or a fragment thereof) in the test sample. Ifthe test sample is from a patient, the method can further comprisediagnosing, prognosticating, or assessing the efficacy oftherapeutic/prophylactic treatment of the patient. If the method furthercomprises assessing the efficacy of therapeutic/prophylactic treatmentof the patient, the method optionally further comprises modifying thetherapeutic/prophylactic treatment of the patient as needed to improveefficacy. The method can be adapted for use in an automated system or asemi-automated system.

Still yet another method of determining the presence, amount orconcentration of an antigen (or a fragment thereof) in a test sample isprovided. The method comprises assaying the test sample for the antigen(or a fragment thereof) by an immunoassay. The immunoassay (i) employsat least one DVD-Ig that can bind two antigens and at least onedetectable label and (ii) comprises comparing a signal generated by thedetectable label as a direct or indirect indication of the presence,amount or concentration of the antigen (or a fragment thereof) in thetest sample to a signal generated as a direct or indirect indication ofthe presence, amount or concentration of the antigen (or a fragmentthereof) in a control or a calibrator. The calibrator is optionally partof a series of calibrators in which each of the calibrators differs fromthe other calibrators in the series by the concentration of the antigen(or a fragment thereof). One of the at least one DVD-Ig (i′) comprisesfour polypeptide chains, wherein the first and third polypeptide chainscomprise a first VD1-(X1)n-VD2-C-(X2)n, in which VD1 is a first heavychain variable domain obtained from a first parent antibody (or antigenbinding portion thereof), VD2 is a second heavy chain variable domainobtained from a second parent antibody (or antigen binding portionthereof), which can be the same as or different from the first parentantibody, C is a heavy chain constant domain, (X1)n is a linker, whichis optionally present and, when present, is other than CH1, and (X2)n isan Fc region, which is optionally present, and wherein the second andfourth polypeptide chains comprise a second VD1-(X1)n-VD2-C-(X2)n, inwhich VD1 is a first light chain variable domain obtained from a firstparent antibody (or antigen binding portion thereof), VD2 is a secondlight chain variable domain obtained from a second parent antibody (orantigen binding portion thereof), which can be the same as or differentfrom the first parent antibody, C is a light chain constant domain,(X1)n is a linker, which is optionally present and, when present, isother than CH1, and (X2)n is an Fc region, which is optionally present,and (ii′) can bind two antigens (or fragments thereof). The method cancomprise (i) contacting the test sample with at least one capture agent,which binds to an epitope on the antigen (or a fragment thereof) so asto form a capture agent/antigen (or a fragment thereof) complex, (ii)contacting the capture agent/antigen (or a fragment thereof) complexwith at least one detection agent, which comprises a detectable labeland binds to an epitope on the antigen (or a fragment thereof) that isnot bound by the capture agent, to form a capture agent/antigen (or afragment thereof)/detection agent complex, and (iii) determining thepresence, amount or concentration of the antigen (or a fragment thereof)in the test sample based on the signal generated by the detectable labelin the capture agent/antigen (or a fragment thereof)/detection agentcomplex formed in (ii), wherein at least one capture agent and/or atleast one detection agent is the at least one DVD-Ig. Alternatively, themethod can comprise (i) contacting the test sample with at least onecapture agent, which binds to an epitope on the antigen (or a fragmentthereof) so as to form a capture agent/antigen (or a fragment thereof)complex, and simultaneously or sequentially, in either order, contactingthe test sample with detectably labeled antigen (or a fragment thereof),which can compete with any antigen (or a fragment thereof) in the testsample for binding to the at least one capture agent, wherein anyantigen (or a fragment thereof) present in the test sample and thedetectably labeled antigen compete with each other to form a captureagent/antigen (or a fragment thereof) complex and a captureagent/detectably labeled antigen (or a fragment thereof) complex,respectively, and (ii) determining the presence, amount or concentrationof the antigen (or a fragment thereof) in the test sample based on thesignal generated by the detectable label in the capture agent/detectablylabeled antigen (or a fragment thereof) complex formed in (ii), whereinat least one capture agent is the at least one DVD-Ig and wherein thesignal generated by the detectable label in the capture agent/detectablylabeled antigen (or a fragment thereof) complex is inverselyproportional to the amount or concentration of antigen (or a fragmentthereof) in the test sample. If the test sample is from a patient, themethod can further comprise diagnosing, prognosticating, or assessingthe efficacy of therapeutic/prophylactic treatment of the patient. Ifthe method further comprises assessing the efficacy oftherapeutic/prophylactic treatment of the patient, the method optionallyfurther comprises modifying the therapeutic/prophylactic treatment ofthe patient as needed to improve efficacy. The method can be adapted foruse in an automated system or a semi-automated system.

With regard to the methods of assay (and kit therefor), it may bepossible to employ commercially available anti-analyte antibodies ormethods for production of anti-analyte as described in the literature.Commercial supplies of various antibodies include, but are not limitedto, Santa Cruz Biotechnology Inc. (Santa Cruz, Calif.), GenWay Biotech,Inc. (San Diego, Calif.), and R&D Systems (RDS; Minneapolis, Minn.).

Generally, a predetermined level can be employed as a benchmark againstwhich to assess results obtained upon assaying a test sample for analyteor a fragment thereof, e.g., for detecting disease or risk of disease.Generally, in making such a comparison, the predetermined level isobtained by running a particular assay a sufficient number of times andunder appropriate conditions such that a linkage or association ofanalyte presence, amount or concentration with a particular stage orendpoint of a disease, disorder or condition or with particular clinicalindicia can be made. Typically, the predetermined level is obtained withassays of reference subjects (or populations of subjects). The analytemeasured can include fragments thereof, degradation products thereof,and/or enzymatic cleavage products thereof.

In particular, with respect to a predetermined level as employed formonitoring disease progression and/or treatment, the amount orconcentration of analyte or a fragment thereof may be “unchanged,”“favorable” (or “favorably altered”), or “unfavorable” (or “unfavorablyaltered”). “Elevated” or “increased” refers to an amount or aconcentration in a test sample that is higher than a typical or normallevel or range (e.g., predetermined level), or is higher than anotherreference level or range (e.g., earlier or baseline sample). The term“lowered” or “reduced” refers to an amount or a concentration in a testsample that is lower than a typical or normal level or range (e.g.,predetermined level), or is lower than another reference level or range(e.g., earlier or baseline sample). The term “altered” refers to anamount or a concentration in a sample that is altered (increased ordecreased) over a typical or normal level or range (e.g., predeterminedlevel), or over another reference level or range (e.g., earlier orbaseline sample).

The typical or normal level or range for analyte is defined inaccordance with standard practice. Because the levels of analyte in someinstances will be very low, a so-called altered level or alteration canbe considered to have occurred when there is any net change as comparedto the typical or normal level or range, or reference level or range,which cannot be explained by experimental error or sample variation.Thus, the level measured in a particular sample will be compared withthe level or range of levels determined in similar samples from aso-called normal subject. In this context, a “normal subject” is anindividual with no detectable disease, for example, and a “normal”(sometimes termed “control”) patient or population is/are one(s) thatexhibit(s) no detectable disease, respectively, for example.Furthermore, given that analyte is not routinely found at a high levelin the majority of the human population, a “normal subject” can beconsidered an individual with no substantial detectable increased orelevated amount or concentration of analyte, and a “normal” (sometimestermed “control”) patient or population is/are one(s) that exhibit(s) nosubstantial detectable increased or elevated amount or concentration ofanalyte. An “apparently normal subject” is one in which analyte has notyet been or currently is being assessed. The level of an analyte is saidto be “elevated” when the analyte is normally undetectable (e.g., thenormal level is zero, or within a range of from about 25 to about 75percentiles of normal populations), but is detected in a test sample, aswell as when the analyte is present in the test sample at a higher thannormal level. Thus, inter alia, the disclosure provides a method ofscreening for a subject having, or at risk of having, a particulardisease, disorder, or condition. The method of assay can also involvethe assay of other markers and the like.

Accordingly, the methods described herein also can be used to determinewhether or not a subject has or is at risk of developing a givendisease, disorder or condition. Specifically, such a method can comprisethe steps of (a) determining the concentration or amount in a testsample from a subject of analyte (or a fragment thereof) (e.g., usingthe methods described herein, or methods known in the art); and (b)comparing the concentration or amount of analyte (or a fragment thereof)determined in step (a) with a predetermined level, wherein, if theconcentration or amount of analyte determined in step (a) is favorablewith respect to a predetermined level, then the subject is determinednot to have or be at risk for a given disease, disorder or condition.However, if the concentration or amount of analyte determined in step(a) is unfavorable with respect to the predetermined level, then thesubject is determined to have or be at risk for a given disease,disorder or condition.

Additionally, provided herein is method of monitoring the progression ofdisease in a subject. Optimally the method comprising the steps of (a)determining the concentration or amount in a test sample from a subjectof analyte; (b) determining the concentration or amount in a later testsample from the subject of analyte; and (c) comparing the concentrationor amount of analyte as determined in step (b) with the concentration oramount of analyte determined in step (a), wherein if the concentrationor amount determined in step (b) is unchanged or is unfavorable whencompared to the concentration or amount of analyte determined in step(a), then the disease in the subject is determined to have continued,progressed or worsened. By comparison, if the concentration or amount ofanalyte as determined in step (b) is favorable when compared to theconcentration or amount of analyte as determined in step (a), then thedisease in the subject is determined to have discontinued, regressed orimproved.

Optionally, the method further comprises comparing the concentration oramount of analyte as determined in step (b), for example, with apredetermined level. Further, optionally the method comprises treatingthe subject with one or more pharmaceutical compositions for a period oftime if the comparison shows that the concentration or amount of analyteas determined in step (b), for example, is unfavorably altered withrespect to the predetermined level.

Still further, the methods can be used to monitor treatment in a subjectreceiving treatment with one or more pharmaceutical compositions.Specifically, such methods involve providing a first test sample from asubject before the subject has been administered one or morepharmaceutical compositions. Next, the concentration or amount in afirst test sample from a subject of analyte is determined (e.g., usingthe methods described herein or as known in the art). After theconcentration or amount of analyte is determined, optionally theconcentration or amount of analyte is then compared with a predeterminedlevel. If the concentration or amount of analyte as determined in thefirst test sample is lower than the predetermined level, then thesubject is not treated with one or more pharmaceutical compositions.However, if the concentration or amount of analyte as determined in thefirst test sample is higher than the predetermined level, then thesubject is treated with one or more pharmaceutical compositions for aperiod of time. The period of time that the subject is treated with theone or more pharmaceutical compositions can be determined by one skilledin the art (for example, the period of time can be from about seven (7)days to about two years, preferably from about fourteen (14) days toabout one (1) year).

During the course of treatment with the one or more pharmaceuticalcompositions, second and subsequent test samples are then obtained fromthe subject. The number of test samples and the time in which said testsamples are obtained from the subject are not critical. For example, asecond test sample could be obtained seven (7) days after the subject isfirst administered the one or more pharmaceutical compositions, a thirdtest sample could be obtained two (2) weeks after the subject is firstadministered the one or more pharmaceutical compositions, a fourth testsample could be obtained three (3) weeks after the subject is firstadministered the one or more pharmaceutical compositions, a fifth testsample could be obtained four (4) weeks after the subject is firstadministered the one or more pharmaceutical compositions, etc.

After each second or subsequent test sample is obtained from thesubject, the concentration or amount of analyte is determined in thesecond or subsequent test sample is determined (e.g., using the methodsdescribed herein or as known in the art). The concentration or amount ofanalyte as determined in each of the second and subsequent test samplesis then compared with the concentration or amount of analyte asdetermined in the first test sample (e.g., the test sample that wasoriginally optionally compared to the predetermined level). If theconcentration or amount of analyte as determined in step (c) isfavorable when compared to the concentration or amount of analyte asdetermined in step (a), then the disease in the subject is determined tohave discontinued, regressed or improved, and the subject shouldcontinue to be administered the one or pharmaceutical compositions ofstep (b). However, if the concentration or amount determined in step (c)is unchanged or is unfavorable when compared to the concentration oramount of analyte as determined in step (a), then the disease in thesubject is determined to have continued, progressed or worsened, and thesubject should be treated with a higher concentration of the one or morepharmaceutical compositions administered to the subject in step (b) orthe subject should be treated with one or more pharmaceuticalcompositions that are different from the one or more pharmaceuticalcompositions administered to the subject in step (b). Specifically, thesubject can be treated with one or more pharmaceutical compositions thatare different from the one or more pharmaceutical compositions that thesubject had previously received to decrease or lower said subject'sanalyte level.

Generally, for assays in which repeat testing may be done (e.g.,monitoring disease progression and/or response to treatment), a secondor subsequent test sample is obtained at a period in time after thefirst test sample has been obtained from the subject. Specifically, asecond test sample from the subject can be obtained minutes, hours,days, weeks or years after the first test sample has been obtained fromthe subject. For example, the second test sample can be obtained fromthe subject at a time period of about 1 minute, about 5 minutes, about10 minutes, about 15 minutes, about 30 minutes, about 45 minutes, about60 minutes, about 2 hours, about 3 hours, about 4 hours, about 5 hours,about 6 hours, about 7 hours, about 8 hours, about 9 hours, about 10hours, about 11 hours, about 12 hours, about 13 hours, about 14 hours,about 15 hours, about 16 hours, about 17 hours, about 18 hours, about 19hours, about 20 hours, about 21 hours, about 22 hours, about 23 hours,about 24 hours, about 2 days, about 3 days, about 4 days, about 5 days,about 6 days, about 7 days, about 2 weeks, about 3 weeks, about 4 weeks,about 5 weeks, about 6 weeks, about 7 weeks, about 8 weeks, about 9weeks, about 10 weeks, about 11 weeks, about 12 weeks, about 13 weeks,about 14 weeks, about 15 weeks, about 16 weeks, about 17 weeks, about 18weeks, about 19 weeks, about 20 weeks, about 21 weeks, about 22 weeks,about 23 weeks, about 24 weeks, about 25 weeks, about 26 weeks, about 27weeks, about 28 weeks, about 29 weeks, about 30 weeks, about 31 weeks,about 32 weeks, about 33 weeks, about 34 weeks, about 35 weeks, about 36weeks, about 37 weeks, about 38 weeks, about 39 weeks, about 40 weeks,about 41 weeks, about 42 weeks, about 43 weeks, about 44 weeks, about 45weeks, about 46 weeks, about 47 weeks, about 48 weeks, about 49 weeks,about 50 weeks, about 51 weeks, about 52 weeks, about 1.5 years, about 2years, about 2.5 years, about 3.0 years, about 3.5 years, about 4.0years, about 4.5 years, about 5.0 years, about 5.5. years, about 6.0years, about 6.5 years, about 7.0 years, about 7.5 years, about 8.0years, about 8.5 years, about 9.0 years, about 9.5 years or about 10.0years after the first test sample from the subject is obtained.

When used to monitor disease progression, the above assay can be used tomonitor the progression of disease in subjects suffering from acuteconditions. Acute conditions, also known as critical care conditions,refer to acute, life-threatening diseases or other critical medicalconditions involving, for example, the cardiovascular system orexcretory system. Typically, critical care conditions refer to thoseconditions requiring acute medical intervention in a hospital-basedsetting (including, but not limited to, the emergency room, intensivecare unit, trauma center, or other emergent care setting) oradministration by a paramedic or other field-based medical personnel.For critical care conditions, repeat monitoring is generally done withina shorter time frame, namely, minutes, hours or days (e.g., about 1minute, about 5 minutes, about 10 minutes, about 15 minutes, about 30minutes, about 45 minutes, about 60 minutes, about 2 hours, about 3hours, about 4 hours, about 5 hours, about 6 hours, about 7 hours, about8 hours, about 9 hours, about 10 hours, about 11 hours, about 12 hours,about 13 hours, about 14 hours, about 15 hours, about 16 hours, about 17hours, about 18 hours, about 19 hours, about 20 hours, about 21 hours,about 22 hours, about 23 hours, about 24 hours, about 2 days, about 3days, about 4 days, about 5 days, about 6 days or about 7 days), and theinitial assay likewise is generally done within a shorter timeframe,e.g., about minutes, hours or days of the onset of the disease orcondition.

The assays also can be used to monitor the progression of disease insubjects suffering from chronic or non-acute conditions. Non-criticalcare or, non-acute conditions, refers to conditions other than acute,life-threatening disease or other critical medical conditions involving,for example, the cardiovascular system and/or excretory system.Typically, non-acute conditions include those of longer-term or chronicduration. For non-acute conditions, repeat monitoring generally is donewith a longer timeframe, e.g., hours, days, weeks, months or years(e.g., about 1 hour, about 2 hours, about 3 hours, about 4 hours, about5 hours, about 6 hours, about 7 hours, about 8 hours, about 9 hours,about 10 hours, about 11 hours, about 12 hours, about 13 hours, about 14hours, about 15 hours, about 16 hours, about 17 hours, about 18 hours,about 19 hours, about 20 hours, about 21 hours, about 22 hours, about 23hours, about 24 hours, about 2 days, about 3 days, about 4 days, about 5days, about 6 days, about 7 days, about 2 weeks, about 3 weeks, about 4weeks, about 5 weeks, about 6 weeks, about 7 weeks, about 8 weeks, about9 weeks, about 10 weeks, about 11 weeks, about 12 weeks, about 13 weeks,about 14 weeks, about 15 weeks, about 16 weeks, about 17 weeks, about 18weeks, about 19 weeks, about 20 weeks, about 21 weeks, about 22 weeks,about 23 weeks, about 24 weeks, about 25 weeks, about 26 weeks, about 27weeks, about 28 weeks, about 29 weeks, about 30 weeks, about 31 weeks,about 32 weeks, about 33 weeks, about 34 weeks, about 35 weeks, about 36weeks, about 37 weeks, about 38 weeks, about 39 weeks, about 40 weeks,about 41 weeks, about 42 weeks, about 43 weeks, about 44 weeks, about 45weeks, about 46 weeks, about 47 weeks, about 48 weeks, about 49 weeks,about 50 weeks, about 51 weeks, about 52 weeks, about 1.5 years, about 2years, about 2.5 years, about 3.0 years, about 3.5 years, about 4.0years, about 4.5 years, about 5.0 years, about 5.5. years, about 6.0years, about 6.5 years, about 7.0 years, about 7.5 years, about 8.0years, about 8.5 years, about 9.0 years, about 9.5 years or about 10.0years), and the initial assay likewise generally is done within a longertime frame, e.g., about hours, days, months or years of the onset of thedisease or condition.

Furthermore, the above assays can be performed using a first test sampleobtained from a subject where the first test sample is obtained from onesource, such as urine, serum or plasma. Optionally, the above assays canthen be repeated using a second test sample obtained from the subjectwhere the second test sample is obtained from another source. Forexample, if the first test sample was obtained from urine, the secondtest sample can be obtained from serum or plasma. The results obtainedfrom the assays using the first test sample and the second test samplecan be compared. The comparison can be used to assess the status of adisease or condition in the subject.

Moreover, the present disclosure also relates to methods of determiningwhether a subject predisposed to or suffering from a given disease,disorder or condition will benefit from treatment. In particular, thedisclosure relates to analyte companion diagnostic methods and products.Thus, the method of “monitoring the treatment of disease in a subject”as described herein further optimally also can encompass selecting oridentifying candidates for therapy.

Thus, in particular embodiments, the disclosure also provides a methodof determining whether a subject having, or at risk for, a givendisease, disorder or condition is a candidate for therapy. Generally,the subject is one who has experienced some symptom of a given disease,disorder or condition or who has actually been diagnosed as having, orbeing at risk for, a given disease, disorder or condition, and/or whodemonstrates an unfavorable concentration or amount of analyte or afragment thereof, as described herein.

The method optionally comprises an assay as described herein, whereanalyte is assessed before and following treatment of a subject with oneor more pharmaceutical compositions (e.g., particularly with apharmaceutical related to a mechanism of action involving analyte), withimmunosuppressive therapy, or by immunoabsorption therapy, or whereanalyte is assessed following such treatment and the concentration orthe amount of analyte is compared against a predetermined level. Anunfavorable concentration of amount of analyte observed followingtreatment confirms that the subject will not benefit from receivingfurther or continued treatment, whereas a favorable concentration oramount of analyte observed following treatment confirms that the subjectwill benefit from receiving further or continued treatment. Thisconfirmation assists with management of clinical studies, and provisionof improved patient care.

It goes without saying that, while certain embodiments herein areadvantageous when employed to assess a given disease, disorder orcondition as discussed herein, the assays and kits can be employed toassess analyte in other diseases, disorders and conditions. The methodof assay can also involve the assay of other markers and the like.

The method of assay also can be used to identify a compound thatameliorates a given disease, disorder or condition. For example, a cellthat expresses analyte can be contacted with a candidate compound. Thelevel of expression of analyte in the cell contacted with the compoundcan be compared to that in a control cell using the method of assaydescribed herein.

II. Kit

A kit for assaying a test sample for the presence, amount orconcentration of an analyte (or a fragment thereof) in a test sample isalso provided. The kit comprises at least one component for assaying thetest sample for the analyte (or a fragment thereof) and instructions forassaying the test sample for the analyte (or a fragment thereof). The atleast one component for assaying the test sample for the analyte (or afragment thereof) can include a composition comprising an anti-analyteDVD-Ig (or a fragment, a variant, or a fragment of a variant thereof),which is optionally immobilized on a solid phase.

The kit can comprise at least one component for assaying the test samplefor an analyte by immunoassay, e.g., chemiluminescent microparticleimmunoassay, and instructions for assaying the test sample for ananalyte by immunoassay, e.g., chemiluminescent microparticleimmunoassay. For example, the kit can comprise at least one specificbinding partner for an analyte, such as an anti-analyte,monoclonal/polyclonal antibody (or a fragment thereof that can bind tothe analyte, a variant thereof that can bind to the analyte, or afragment of a variant that can bind to the analyte) or an anti-analyteDVD-Ig (or a fragment, a variant, or a fragment of a variant thereof),either of which can be detectably labeled. Alternatively oradditionally, the kit can comprise detectably labeled analyte (or afragment thereof that can bind to an anti-analyte, monoclonal/polyclonalantibody or an anti-analyte DVD-Ig (or a fragment, a variant, or afragment of a variant thereof)), which can compete with any analyte in atest sample for binding to an anti-analyte, monoclonal/polyclonalantibody (or a fragment thereof that can bind to the analyte, a variantthereof that can bind to the analyte, or a fragment of a variant thatcan bind to the analyte) or an anti-analyte DVD-Ig (or a fragment, avariant, or a fragment of a variant thereof), either of which can beimmobilized on a solid support. The kit can comprise a calibrator orcontrol, e.g., isolated or purified analyte. The kit can comprise atleast one container (e.g., tube, microtiter plates or strips, which canbe already coated with a first specific binding partner, for example)for conducting the assay, and/or a buffer, such as an assay buffer or awash buffer, either one of which can be provided as a concentratedsolution, a substrate solution for the detectable label (e.g., anenzymatic label), or a stop solution. Preferably, the kit comprises allcomponents, i.e., reagents, standards, buffers, diluents, etc., whichare necessary to perform the assay. The instructions can be in paperform or computer-readable form, such as a disk, CD, DVD, or the like.

More specifically, provided is a kit for assaying a test sample for anantigen (or a fragment thereof). The kit comprises at least onecomponent for assaying the test sample for an antigen (or a fragmentthereof) and instructions for assaying the test sample for an antigen(or a fragment thereof), wherein the at least one component includes atleast one composition comprising a binding protein, which (i′) comprisesa polypeptide chain comprising VD1-(X1)n-VD2-C-(X2)n, in which VD1 is afirst heavy chain variable domain obtained from a first parent antibody(or antigen binding portion thereof), VD2 is a second heavy chainvariable domain obtained from a second parent antibody (or antigenbinding portion thereof), which can be same as or different from thefirst parent antibody, C is a heavy chain constant domain, (X1)n is alinker, which is optionally present and, when present, is other thanCH1, and (X2)n is an Fc region, which is optionally present, and (ii′)can bind a pair of antigens, wherein the binding protein is optionallydetectably labeled.

Further provided is another kit for assaying a test sample for anantigen (or a fragment thereof). The kit comprises at least onecomponent for assaying the test sample for an antigen (or a fragmentthereof) and instructions for assaying the test sample for an antigen(or a fragment thereof), wherein the at least one component includes atleast one composition comprising a binding protein, which (i′) comprisesa polypeptide chain comprising VD1-(X1)n-VD2-C-(X2)n, in which VD1 is afirst light chain variable domain obtained from a first parent antibody(or antigen binding portion thereof), VD2 is a second light chainvariable domain obtained from a second parent antibody (or antigenbinding portion thereof), which can be the same as or different from thefirst parent antibody, C is a light chain constant domain, (X1)n is alinker, which is optionally present and, when present, is other thanCH1, and (X2)n is an Fc region, which is optionally present, and (ii′)can bind a pair of antigens, wherein the binding protein is optionallydetectably labeled.

Still further provided is another kit for assaying a test sample for anantigen (or a fragment thereof). The kit comprises at least onecomponent for assaying the test sample for an antigen (or a fragmentthereof) and instructions for assaying the test sample for an antigen(or a fragment thereof), wherein the at least one component includes atleast one composition comprising a binding protein, which (i′) comprisesa first polypeptide chain and a second polypeptide chain, wherein thefirst polypeptide chain comprises a first VD1-(X1)n-VD2-C-(X2)n, inwhich VD1 is a first heavy chain variable domain obtained from a firstparent antibody (or antigen binding portion thereof), VD2 is a secondheavy chain variable domain obtained from a second parent antibody (orantigen binding portion thereof), which can be the same as or differentfrom the first parent antibody, C is a heavy chain constant domain,(X1)n is a linker, which is optionally present and, when present, isother than CH1, and (X2)n is an Fc region, which is optionally present,and wherein the second polypeptide chain comprises a secondVD1-(X1)n-VD2-C-(X2)n, in which VD1 is a first light chain variabledomain obtained from a first parent antibody (or antigen binding portionthereof), VD2 is a second light chain variable domain obtained from asecond parent antibody (or antigen binding portion thereof), which canbe the same as or different from the first parent antibody, C is a lightchain constant domain, (X1)n is a linker, which is optionally presentand, when present, is other than CH1, and (X2)n is an Fc region, whichis optionally present, and (ii′) can bind a pair of antigens, whereinthe binding protein is optionally detectably labeled.

Even still further provided is another kit for assaying a test samplefor an antigen (or a fragment thereof). The kit comprises at least onecomponent for assaying the test sample for an antigen (or a fragmentthereof) and instructions for assaying the test sample for an antigen(or a fragment thereof), wherein the at least one component includes atleast one composition comprising a DVD-Ig, which (i′) comprises fourpolypeptide chains, wherein the first and third polypeptide chainscomprise a first VD1-(X1)n-VD2-C-(X2)n, in which VD1 is a first heavychain variable domain obtained from a first parent antibody (or antigenbinding portion thereof), VD2 is a second heavy chain variable domainobtained from a second parent antibody (or antigen binding portionthereof), which can be the same as or different from the first parentantibody, C is a heavy chain constant domain, (X1)n is a linker, whichis optionally present and, when present, is other than CH1, and (X2)n isan Fc region, which is optionally present, and wherein the second andfourth polypeptide chains comprise a second VD1-(X1)n-VD2-C-(X2)n, inwhich VD1 is a first light chain variable domain obtained from a firstparent antibody (or antigen binding portion thereof), VD2 is a secondlight chain variable domain obtained from a second parent antibody (orantigen binding portion thereof), which can be the same as or differentfrom the first parent antibody, C is a light chain constant domain,(X1)n is a linker, which is optionally present and, when present, isother than CH1, and (X2)n is an Fc region, which is optionally present,and (ii′) can bind two antigens (or fragments thereof), wherein theDVD-Ig is optionally detectably labeled.

Any antibodies, such as an anti-analyte antibody or an anti-analyteDVD-Ig, or tracer can incorporate a detectable label, such as afluorophore, a radioactive moiety, an enzyme, a biotin/avidin label, achromophore, a chemiluminescent label, or the like, or the kit caninclude reagents for carrying out detectable labeling. The antibodies,calibrators and/or controls can be provided in separate containers orpre-dispensed into an appropriate assay format, for example, intomicrotiter plates.

Optionally, the kit includes quality control components (for example,sensitivity panels, calibrators, and positive controls). Preparation ofquality control reagents is well-known in the art and is described oninsert sheets for a variety of immunodiagnostic products. Sensitivitypanel members optionally are used to establish assay performancecharacteristics, and further optionally are useful indicators of theintegrity of the immunoassay kit reagents, and the standardization ofassays.

The kit can also optionally include other reagents required to conduct adiagnostic assay or facilitate quality control evaluations, such asbuffers, salts, enzymes, enzyme co-factors, enzyme substrates, detectionreagents, and the like. Other components, such as buffers and solutionsfor the isolation and/or treatment of a test sample (e.g., pretreatmentreagents), also can be included in the kit. The kit can additionallyinclude one or more other controls. One or more of the components of thekit can be lyophilized, in which case the kit can further comprisereagents suitable for the reconstitution of the lyophilized components.

The various components of the kit optionally are provided in suitablecontainers as necessary, e.g., a microtiter plate. The kit can furtherinclude containers for holding or storing a sample (e.g., a container orcartridge for a urine sample). Where appropriate, the kit optionallyalso can contain reaction vessels, mixing vessels, and other componentsthat facilitate the preparation of reagents or the test sample. The kitcan also include one or more instruments for assisting with obtaining atest sample, such as a syringe, pipette, forceps, measured spoon, or thelike.

If the detectable label is at least one acridinium compound, the kit cancomprise at least one acridinium-9-carboxamide, at least oneacridinium-9-carboxylate aryl ester, or any combination thereof. If thedetectable label is at least one acridinium compound, the kit also cancomprise a source of hydrogen peroxide, such as a buffer, a solution,and/or at least one basic solution. If desired, the kit can contain asolid phase, such as a magnetic particle, bead, test tube, microtiterplate, cuvette, membrane, scaffolding molecule, film, filter paper, discor chip.

C. Adaptation of Kit and Method

The kit (or components thereof), as well as the method of determiningthe presence, amount or concentration of an analyte in a test sample byan assay, such as an immunoassay can be adapted for use in a variety ofautomated and semi-automated systems (including those wherein the solidphase comprises a microparticle), as described, e.g., in U.S. Pat. Nos.5,089,424 and 5,006,309, and as commercially marketed, e.g., by AbbottLaboratories (Abbott Park, Ill.) as ARCHITECT®.

Some of the differences between an automated or semi-automated system ascompared to a non-automated system (e.g., ELISA) include the substrateto which the first specific binding partner (e.g., an anti-analyte,monoclonal/polyclonal antibody (or a fragment thereof, a variantthereof, or a fragment of a variant thereof) or an anti-analyte DVD-Ig(or a fragment thereof, a variant thereof, or a fragment of a variantthereof) is attached; either way, sandwich formation and analytereactivity can be impacted), and the length and timing of the capture,detection and/or any optional wash steps. Whereas a non-automatedformat, such as an ELISA, may require a relatively longer incubationtime with sample and capture reagent (e.g., about 2 hours), an automatedor semi-automated format (e.g., ARCHITECT®, Abbott Laboratories) mayhave a relatively shorter incubation time (e.g., approximately 18minutes for ARCHITECT®). Similarly, whereas a non-automated format, suchas an ELISA, may incubate a detection antibody, such as the conjugatereagent, for a relatively longer incubation time (e.g., about 2 hours),an automated or semi-automated format (e.g., ARCHITECT® may have arelatively shorter incubation time (e.g., approximately 4 minutes forthe ARCHITECT®).

Other platforms available from Abbott Laboratories include, but are notlimited to, AxSYM®, IMx® (U.S. Pat. No. 5,294,404), PRISM®, EIA (bead),and Quantum™ II, as well as other platforms. Additionally, the assays,kits and kit components can be employed in other formats, for example,on electrochemical or other hand-held or point-of-care assay systems.The present disclosure is, for example, applicable to the commercialAbbott Point of Care (i-STAT®, Abbott Laboratories) electrochemicalimmunoassay system that performs sandwich immunoassays. Immunosensorsand their methods of manufacture and operation in single-use testdevices are described, for example in, U.S. Pat. Nos. 5,063,081;7,419,821; and 7,682,833; and U.S. Patent Publication Nos. 20040018577and 20060160164.

In particular, with regard to the adaptation of an analyte assay to theI-STAT® system, the following configuration is preferred. Amicrofabricated silicon chip is manufactured with a pair of goldamperometric working electrodes and a silver-silver chloride referenceelectrode. On one of the working electrodes, polystyrene beads (0.2 mmdiameter) with immobilized anti-analyte, monoclonal/polyclonal antibody(or a fragment thereof, a variant thereof, or a fragment of a variantthereof) or anti-analyte DVD-Ig (or a fragment thereof, a variantthereof, or a fragment of a variant thereof), are adhered to a polymercoating of patterned polyvinyl alcohol over the electrode. This chip isassembled into an I-STAT® cartridge with a fluidics format suitable forimmunoassay. On a portion of the wall of the sample-holding chamber ofthe cartridge there is a layer comprising a specific binding partner foran analyte, such as an anti-analyte, monoclonal/polyclonal antibody (ora fragment thereof, a variant thereof, or a fragment of a variantthereof that can bind the analyte) or an anti-analyte DVD-Ig (or afragment thereof, a variant thereof, or a fragment of a variant thereofthat can bind the analyte), either of which can be detectably labeled.Within the fluid pouch of the cartridge is an aqueous reagent thatincludes p-aminophenol phosphate.

In operation, a sample suspected of containing an analyte is added tothe holding chamber of the test cartridge, and the cartridge is insertedinto the I-STAT® reader. After the specific binding partner for ananalyte has dissolved into the sample, a pump element within thecartridge forces the sample into a conduit containing the chip. Here itis oscillated to promote formation of the sandwich. In the penultimatestep of the assay, fluid is forced out of the pouch and into the conduitto wash the sample off the chip and into a waste chamber. In the finalstep of the assay, the alkaline phosphatase label reacts withp-aminophenol phosphate to cleave the phosphate group and permit theliberated p-aminophenol to be electrochemically oxidized at the workingelectrode. Based on the measured current, the reader is able tocalculate the amount of analyte in the sample by means of an embeddedalgorithm and factory-determined calibration curve.

The methods and kits as described herein necessarily encompass otherreagents and methods for carrying out the immunoassay. For instance,encompassed are various buffers such as are known in the art and/orwhich can be readily prepared or optimized to be employed, e.g., forwashing, as a conjugate diluent, microparticle diluent, and/or as acalibrator diluent. An exemplary conjugate diluent is ARCHITECT®conjugate diluent employed in certain kits (Abbott Laboratories, AbbottPark, Ill.) and containing 2-(N-morpholino)ethanesulfonic acid (MES), asalt, a protein blocker, an antimicrobial agent, and a detergent. Anexemplary calibrator diluent is ARCHITECT® human calibrator diluentemployed in certain kits (Abbott Laboratories, Abbott Park, Ill.), whichcomprises a buffer containing MES, other salt, a protein blocker, and anantimicrobial agent. Additionally, as described in U.S. PatentApplication No. 61/142,048 filed Dec. 31, 2008, improved signalgeneration may be obtained, e.g., in an I-Stat cartridge format, using anucleic acid sequence linked to the signal antibody as a signalamplifier.

EXEMPLIFICATION Example 1 Design, Construction, and Analysis of aDVD-Ig™ Molecule Example 1.1 Assays Used to Identify and CharacterizeParent Antibodies and DVD-Ig Molecules

The following assays were used throughout the Examples to identify andcharacterize parent antibodies and DVD-Ig molecules, unless otherwisestated.

Example 1.1.1 Assays Used to Determine Binding and Affinity of ParentAntibodies and DVD-Ig Molecules for their Target Antigen(s) Example1.1.1A Direct Bind ELISA

Enzyme Linked Immunosorbent Assays to screen for antibodies that bind adesired target antigen are performed as follows. High bind ELISA plates(Corning Costar #3369, Acton, Mass.) are coated with 100 μL/well of 10μg/ml of desired target antigen (R&D Systems, Minneapolis, Minn.) ordesired target antigen extra-cellular domain/FC fusion protein (R&DSystems, Minneapolis, Minn.) or monoclonal mouse anti-polyHistidineantibody (R&D Systems #MAB050, Minneapolis, Minn.) in phosphate bufferedsaline (10×PBS, Abbott Bioresearch Center, Media Prep #MPS-073,Worcester, Mass.) overnight at 4° C. Plates are washed four times withPBS containing 0.02% Tween 20. Plates are blocked by the addition of 300μL/well blocking solution (non-fat dry milk powder, various retailsuppliers, diluted to 2% in PBS) for ½ hour at room temperature. Platesare washed four times after blocking with PBS containing 0.02% Tween 20.

Alternatively, one hundred microliters per well of 10 μg/ml of Histidine(H is) tagged desired target antigen (R&D Systems, Minneapolis, Minn.)is added to ELISA plates coated with monoclonal mouse anti-polyHistidineantibody as described above and incubated for 1 hour at roomtemperature. Wells are washed four times with PBS containing 0.02% Tween20.

One hundred microliters of antibody or DVD-Ig preparations diluted inblocking solution as described above is added to the desired targetantigen plate or desired target antigen/FC fusion plate or theanti-polyHistidine antibody/Histidine tagged desired target antigenplate prepared as described above and incubated for 1 hour at roomtemperature. Wells are washed four times with PBS containing 0.02% Tween20.

One hundred microliters of 10 ng/mL goat anti-human IgG-FC specific HRPconjugated antibody (Southern Biotech #2040-05, Birmingham, Ala.) isadded to each well of the desired target antigen plate oranti-polyHistidine antibody/Histidine tagged desired target antigenplate. Alternatively, one hundred microliters of 10 ng/mL goatanti-human IgG-kappa light chain specific HRP conjugated antibody(Southern Biotech #2060-05 Birmingham, Ala.) is added to each well ofthe desired target antigen/FC fusion plate and incubated for 1 hour atroom temperature. Plates are washed 4 times with PBS containing 0.02%Tween 20.

One hundred microliters of enhanced TMB solution (Neogen Corp. #308177,K Blue, Lexington, Ky.) is added to each well and incubated for 10minutes at room temperature. The reaction is stopped by the addition of50 μL 1N sulphuric acid. Plates are read spectrophotometrically at awavelength of 450 nm.

In the Direct Bind ELISA, binding is sometimes not observed, probablybecause the antibody binding site on the target antigen is either“masked” or the antigen is “distorted” when coated to the plasticsurface. The inability of a DVD-Ig molecule to bind its target may alsobe due to steric limitation imposed on DVD-Ig molecules by the DirectBind ELISA format. The parent antibodies and DVD-Ig molecules that donot bind in the Direct Bind ELISA format can bind to target antigen inother ELISA formats, such as FACS, Biacore, or bioassay. Non-binding ofa DVD-Ig molecule is also restored by adjusting the linker lengthbetween the two variable domains of the DVD-Ig molecule, as shownpreviously.

Example 1.1.1.B Capture ELISA

To determine the binding of parental and DVD-Ig proteins to IL-1α orIL-1β by capture ELISA, ELISA plates (Nunc, MaxiSorp, Rochester, N.Y.)were incubated overnight at 4° C. with anti-human Fc antibody diluted inPierce Coat buffer at 2 μg/ml (Jackson Immunoresearch, West Grove, Pa.).Plates were washed five times in washing buffer (PBS containing 0.05%Tween 20) and blocked for 1 hour at 25° C. with 200 μl per wellsuperblock blocking buffer (Thermo scientific, #37515). Blocking bufferwas removed, and 2 μg/ml of each antibody or DVD-Ig molecule in PBScontaining 10% superblock, 0.5% Tween-20 was added to the wells at 100μl per well and incubated at 25° C. for 1 hour. The wells were washedfive times in 1×PBST, and 1 μg/ml biotinylated antigen was titrated at1:6 serial dilutions (for a range of μg to pg in PBS containing 10%superblock, 0.05% tween 20). Each dilution of antigen was then added tothe plates and incubated for 1 hour at 25° C. The wells were washed fivetimes in 1×PBST and incubated for 1 hour at 25° C. with polyHRPstreptavidin (KPL #474-3000, Gaithersburg, Md.). The wells were washedfive times in 1×PBST, and 100 μl of ULTRA-TMB ELISA (Pierce, Rockford,Ill.) were added per well. Following color development the reaction wasstopped with 1N HCL and absorbance at 450 nM is measured. Table 3 showscapture ELISA data for DVD-Ig constructs targeting IL-1α and IL-1β, andthe numerical value indicates binding of human anti-IL-1α, anti-IL-1βantibodies, or anti-IL-1α/β DVD-Ig proteins to human IL-1α and/or IL-1β.

TABLE 3 Capture ELISA of Parental Antibodies and IL-1α and IL-1β DVD-IgConstructs Capture Capture Parent N-terminal C-terminal ELISA ELISAAntibody or Variable Variable N-terminal VD C-terminal VD DVD-Ig IDDomain Domain EC50 (pM) EC50 (pM) E26.13 IL-1β 7.7 3D12.8 IL-1α 283D12.16 IL-1α 10 3D12-362-10/ IL-1α 7.0 372-15 3D12r16 IL-1α 72.0 E26.35IL-1β 10.4 E26.35-SS- IL-1β IL-1α 6.3 10.6 3D12r16 1B12.1 IL-1β 39.91B12.1-SS- IL-1β IL-1α 40.9 8.7 3D12r16 1B12.3 IL-1β 43.8 1B12.3-SS-IL-1β IL-1α 35.2 20.8 3D12r16 1B12.6 IL-1β 45.9 1B12.6-SS- IL-1β IL-1α65.1 9.9 3D12r16

Example 1.1.1.C Affinity Determination Using BIACORE Technology

TABLE 4 Reagent Used in Biacore Analyses Antigen Vendor DesignationVendor Catalog # IL-1α Recombinant Human IL-1α R&D 200-LA/CF systemsIL-1β Recombinant Human IL-1β R&D 201-LB systemsBIACORE Methods:

The BIACORE assay (Biacore, Inc, Piscataway, N.J.) determines theaffinity of antibodies or DVD-Ig molecule with kinetic measurements ofon-rate and off-rate constants. Binding of antibodies or DVD-Ig moleculeto a target antigen (for example, a purified recombinant target antigen)is determined by surface plasmon resonance-based measurements with aBiacore® 1000 or 3000 instrument (Biacore® AB, Uppsala, Sweden) usingrunning HBS-EP (10 mM HEPES [pH 7.4], 150 mM NaCl, 3 mM EDTA, and 0.005%surfactant P20) at 25° C. All chemicals are obtained from Biacore® AB(Uppsala, Sweden) or otherwise from a different source as described inthe text. For example, approximately 5000 RU of goat anti-mouse IgG,(Fcγ), fragment specific polyclonal antibody (Pierce Biotechnology Inc,Rockford, Ill.) diluted in 10 mM sodium acetate (pH 4.5) is directlyimmobilized across a CM5 research grade biosensor chip using a standardamine coupling kit according to manufacturer's instructions andprocedures at 25 μg/ml. Unreacted moieties on the biosensor surface areblocked with ethanolamine. Modified carboxymethyl dextran surface inflowcell 2 and 4 is used as a reaction surface. Unmodified carboxymethyldextran without goat anti-mouse IgG in flow cell 1 and 3 is used as thereference surface. For kinetic analysis, rate equations derived from the1:1 Langmuir binding model are fitted simultaneously to association anddissociation phases of all eight injections (using global fit analysis)with the use of Biaevaluation 4.0.1 software. Purified antibodies orDVD-Ig molecule are diluted in HEPES-buffered saline for capture acrossgoat anti-mouse IgG specific reaction surfaces. Antibodies or DVD-Igmolecule to be captured as a ligand (25 μg/ml) are injected overreaction matrices at a flow rate of 5 μl/min. The association anddissociation rate constants, k_(on) (M⁻¹s⁻¹) and k_(off) (s⁻¹) aredetermined under a continuous flow rate of 25 μl/minute. Rate constantsare derived by making kinetic binding measurements at different antigenconcentrations ranging from 10-200 nM. The equilibrium dissociationconstant (M) of the reaction between antibodies or DVD-Ig molecules andthe target antigen is then calculated from the kinetic rate constants bythe following formula: K_(D)=k_(off)/k_(on). Binding is recorded as afunction of time and kinetic rate constants are calculated. In thisassay, on-rates as fast as 10⁶M⁻¹s⁻¹ and off-rates as slow as 10⁻⁶ s⁻¹can be measured.

TABLE 5 BIACORE Analysis of Parental Antibodies and DVD-Ig ConstructsN-Terminal C-Terminal Variable Variable Parent Antibody Domain Domaink_(on) k_(off) K_(D) or DVD-Ig ID (VD) (VD) (M-1s-1) (s-1) (M) E26.13IL-1β  9.5 × 10⁺⁵ 4.23 × 10⁻⁵ 4.45 × 10⁻¹¹ E26.35 IL-1β 1.02 × 10⁺⁶ 2.50× 10⁻⁵ 2.39 × 10⁻¹¹ 1B12.1 IL-1β 5.55 × 10⁺⁶ 1.03 × 10⁻³ 1.86 × 10⁻¹⁰1B12.3 IL-1β 5.73 × 10⁺⁶ 1.18 × 10⁻³ 2.06 × 10⁻¹⁰ 1B12.6 IL-1β 6.20 ×10⁺⁶ 1.25 × 10⁻³ 2.02 × 10⁻¹⁰ 3D12.8 IL-1α 2.04 × 10⁺⁶ 2.74 × 10⁻³ 1.35× 10⁻⁹  3D12.16 IL-1α 1.21 × 10⁺⁶ 2.53 × 10⁻³ 2.09 × 10⁻⁹  3D12r16 IL-1α  9 × 10⁺⁵ 2.77 × 10⁻⁵  3.1 × 10⁻¹¹ 3D12.10/15 IL-1α 3.55 × 10⁺⁵ 4.10 ×10⁻⁵ 1.15 × 10⁻¹⁰ E26.13-SS- IL-1α 5.69 × 10⁺⁴ 3.25 × 10⁻⁵ 5.69 × 10⁻¹⁰3D12.10/15 E26.13-SS- IL-1β 1.66 × 10⁺⁶ 2.65 × 10⁻⁵ 1.61 × 10⁻¹¹3D12.10/15 E26.35-SS- IL-1α 5.81 × 10⁺⁴ 2.34 × 10⁻⁵ 4.03 × 10⁻¹⁰3D12.10/15 E26.35-SS- IL-1β 1.28 × 10⁺⁶ 7.85 × 10⁻⁶ 6.11 × 10⁻¹²3D12.10/15 E26.35-SS- IL-1α 9.36 × 10⁺⁴   <1 × 10⁻⁶   <1 × 10⁻¹¹ 3D12r16E26.35-SS- IL-1β 1.17 × 10⁺⁶ 4.11 × 10⁻⁶ 3.52 × 10⁻¹² 3D12r16 1B12.1-SS-IL-1α 1.11 × 10⁺⁵ 1.33 × 10⁻⁵  1.2 × 10⁻¹⁰ 3D12r16 1B12.1-SS- IL-1β 3.08× 10⁺⁶ 1.10 × 10⁻³ 3.55 × 10⁻¹⁰ 3D12r16 1B12.3-SS- IL-1α 4.39 × 10⁺⁴  <1 × 10⁻⁶ <2.30 × 10⁻¹¹   3D12r16 1B12.3-SS- IL-1β 3.70 × 10⁺⁶ 1.17 ×10⁻³ 3.15 × 10⁻¹⁰ 3D12r16 1B12.6-SS- IL-1α 5.62 × 10⁺⁴   <1 × 10⁻⁶ <1.78× 10⁻¹¹   3D12r16 1B12.6-SS- IL-1β 2.93 × 10⁺⁶ 1.84 × 10⁻³ 6.26 × 10⁻¹⁰3D12r16

Binding of all DVD-Ig constructs characterized by Biacore technology wasmaintained and comparable to that of parent antibodies. N-terminalvariable domains bound with a similar high affinity as the parentantibody.

TABLE 6 BIACORE Analysis of Parental Antibodies and DVD-Ig Constructs onCynomolgus IL-1α and IL-1β N-Ter- C-Ter- Parent minal minal AntibodyVariable Variable or DVD-Ig Domain Domain k_(on) k_(off) K_(D) ID (VD)(VD) (M-1s-1) (s-1) (M) E26.13 IL-1β 1.23 × 10⁺⁶ 1.79 × 10⁻⁵ 1.46 ×10⁻¹¹ E26.35 IL-1β 9.21 × 10⁺⁵ 1.29 × 10⁻⁵  1.4 × 10⁻¹¹ 3D12.10/15 IL-1α3.89 × 10⁺⁵ 2.25 × 10⁻⁵ 5.78 × 10⁻¹¹ E26.35-SS- IL-1α 1.01 × 10⁺⁵ 8.23 ×10⁻⁶ 8.11 × 10⁻¹¹ 3D12r16 E26.35-SS- IL-1β 3.95 × 10⁺⁵ 7.91 × 10⁻⁶  2.0× 10⁻¹¹ 3D12r16

Binding of all DVD-Ig constructs characterized by Biacore technology wasmaintained and comparable to that of parent antibodies. N-terminalvariable domains bound with a similar high affinity as the parentantibody.

Example 1.1.2 Assays Used to Determine the Functional Activity of ParentAntibodies and DVD-Ig Molecule Example 1.1.2.A Cytokine Bioassay

The ability of an anti-cytokine or an anti-growth factor parent antibodyor DVD-Ig molecule containing anti-cytokine or anti-growth factorsequences to inhibit or neutralize a target cytokine or growth factorbioactivity is analyzed by determining the inhibitory potential of theantibody or DVD-Ig molecule. For example, the ability of an anti-IL-4antibody to inhibit IL-4 mediated IgE production may be used. Forexample, human naive B cells are isolated from peripheral blood,respectively, buffy coats by Ficoll-paque density centrifugation,followed by magnetic separation with MACS beads (Miltenyi Biotec,Bergisch Gladbach, Germany) specific for human sIgD FITC labeled goatF(ab)2 antibodies followed by anti-FITC MACS beads. Magnetically sortednaive B cells are adjusted to 3×10⁵ cells per ml in XV15 and plated outin 100 μl per well of 96-well plates in a 6×6 array in the center of theplate, surrounded by PBS filled wells during the 10 days of culture at37° C. in the presence of 5% CO₂. One plate each is prepared perantibody to be tested, consisting of 3 wells each of un-induced andinduced controls and quintuplicate repeats of antibody titrationsstarting at 7 μg/ml and running in 3-fold dilution down to 29 ng/mlfinal concentrations added in 50 μl four times concentratedpre-dilution. To induce IgE production, rhIL-4 at 20 ng/ml plusanti-CD40 monoclonal antibody (Novartis, Basel, Switzerland) at 0.5μg/ml final concentrations in 50 μl each are added to each well, and IgEconcentrations are determined at the end of the culture period by astandard sandwich ELISA method.

Example 1.1.2.B Cytokine Release Assay

The ability of a parent antibody or DVD-Ig molecule to cause cytokinerelease is analyzed. Peripheral blood is withdrawn from three healthydonors by venipuncture into heparized vacutainer tubes. Whole blood isdiluted 1:5 with RPMI-1640 medium and placed in 24-well tissue cultureplates at 0.5 mL per well. The anti-cytokine antibodies (e.g.,anti-IL-4) are diluted into RPMI-1640 and placed in the plates at 0.5mL/well to give final concentrations of 200, 100, 50, 10, and 1 μg/mL.The final dilution of whole blood in the culture plates is 1:10. LPS andPHA are added to separate wells at 2 μg/mL and 5 μg/mL finalconcentration as a positive control for cytokine release. Polyclonalhuman IgG is used as negative control antibody. The experiment isperformed in duplicate. Plates are incubated at 37° C. at 5% CO₂.Twenty-four hours later the contents of the wells are transferred intotest tubes and spun for 5 minutes at 1200 rpm. Cell-free supernatantsare collected and frozen for cytokine assays. Cells left over on theplates and in the tubes are lysed with 0.5 mL of lysis solution, andplaced at −20° C. and thawed. 0.5 mL of medium is added (to bring thevolume to the same level as the cell-free supernatant samples) and thecell preparations are collected and frozen for cytokine assays.Cell-free supernatants and cell lysates are assayed for cytokine levelsby ELISA, for example, for levels of IL-8, IL-6, IL-1β, IL-1RA, orTNF-α.

Example 1.1.2.C Cytokine Cross-Reactivity Study

The ability of an anti-cytokine parent antibody or DVD-Ig moleculedirected to a cytokine(s) of interest to cross react with othercytokines is analyzed. Parent antibodies or DVD-Ig molecule areimmobilized on a Biacore biosensor matrix. An anti-human Fc mAb iscovalently linked via free amine groups to the dextran matrix by firstactivating carboxyl groups on the matrix with 100 mMN-hydroxysuccinimide (NHS) and 400 mMN-Ethyl-N′-(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC).Approximately 50 μL of each antibody or DVD-Ig preparation at aconcentration of 25 μg/mL, diluted in sodium acetate, pH 4.5, isinjected across the activated biosensor and free amines on the proteinare bound directly to the activated carboxyl groups. Typically, 5000Resonance Units (RU's) are immobilized. Unreacted matrix EDC-esters aredeactivated by an injection of 1 M ethanolamine. A second flow cell isprepared as a reference standard by immobilizing human IgG1/K using thestandard amine coupling kit. SPR measurements are performed using the CMbiosensor chip. All antigens to be analyzed on the biosensor surface arediluted in HBS-EP running buffer containing 0.01% P20.

To examine the cytokine binding specificity, excess cytokine of interest(100 nM, e.g., soluble recombinant human) is injected across theanti-cytokine parent antibody or DVD-Ig molecule immobilized biosensorsurface (5 minute contact time). Before injection of the cytokine ofinterest and immediately afterward, HBS-EP buffer alone flows througheach flow cell. The net difference in the signals between the baselineand the point corresponding to approximately 30 seconds after completionof cytokine injection are taken to represent the final binding value.Again, the response is measured in Resonance Units. Biosensor matricesare regenerated using 10 mM HCl before injection of the next samplewhere a binding event is observed, otherwise running buffer was injectedover the matrices. Human cytokines (e.g., IL-1α, IL-1β, IL-2, IL-3,IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-15,IL-16, IL-17, IL-18, IL-19, IL-20, IL-22, IL-23, IL-27, TNF-α, TNF-β,and IFN-γ, for example) are also simultaneously injected over theimmobilized mouse IgG1/K reference surface to record any nonspecificbinding background. By preparing a reference and reaction surface,Biacore can automatically subtract the reference surface data from thereaction surface data in order to eliminate the majority of therefractive index change and injection noise. Thus, it is possible toascertain the true binding response attributed to an anti-cytokineantibody or DVD-Ig molecule binding reaction.

When a cytokine of interest is injected across immobilized anti-cytokineantibody, significant binding is observed. 10 mM HCl regenerationcompletely removes all non-covalently associated proteins. Examinationof the sensorgram shows that immobilized anti-cytokine antibody orDVD-Ig molecule binding to soluble cytokine is strong and robust. Afterconfirming the expected result with the cytokine of interest, the panelof remaining recombinant human cytokines is tested, for each antibody orDVD-Ig molecule separately. The amount of anti-cytokine antibody orDVD-Ig molecule bound or unbound cytokine for each injection cycle isrecorded. The results from three independent experiments are used todetermine the specificity profile of each antibody or DVD-Ig molecule.Antibodies or DVD-Ig molecule with the expected binding to the cytokineof interest and no binding to any other cytokine are selected.

Example 1.1.2.D Tissue Cross Reactivity

Tissue cross reactivity studies are done in three stages, with the firststage including cryosections of 32 tissues, second stage including up to38 tissues, and the 3^(rd) stage including additional tissues from 3unrelated adults as described below. Studies are done typically at twodose levels.

Stage 1:

Cryosections (about 5 μm) of human tissues (32 tissues (typically:Adrenal Gland, Gastrointestinal Tract, Prostate, Bladder, Heart,Skeletal Muscle, Blood Cells, Kidney, Skin, Bone Marrow, Liver, SpinalCord, Breast, Lung, Spleen, Cerebellum, Lymph Node, Testes, CerebralCortex, Ovary, Thymus, Colon, Pancreas, Thyroid, Endothelium,Parathyroid, Ureter, Eye, Pituitary, Uterus, Fallopian Tube andPlacenta) from one human donor obtained at autopsy or biopsy) are fixedand dried on object glass. The peroxidase staining of tissue sections isperformed, using the avidin-biotin system.

Stage 2:

Cryosections (about 5 μm) of human tissues 38 tissues (includingadrenal, blood, blood vessel, bone marrow, cerebellum, cerebrum, cervix,esophagus, eye, heart, kidney, large intestine, liver, lung, lymph node,breast mammary gland, ovary, oviduct, pancreas, parathyroid, peripheralnerve, pituitary, placenta, prostate, salivary gland, skin, smallintestine, spinal cord, spleen, stomach, striated muscle, testis,thymus, thyroid, tonsil, ureter, urinary bladder, and uterus) from 3unrelated adults obtained at autopsy or biopsy) are fixed and dried onobject glass. The peroxidase staining of tissue sections is performed,using the avidin-biotin system.

Stage 3:

Cryosections (about 5 μm) of cynomolgus monkey tissues (38 tissues(including adrenal, blood, blood vessel, bone marrow, cerebellum,cerebrum, cervix, esophagus, eye, heart, kidney, large intestine, liver,lung, lymph node, breast mammary gland, ovary, oviduct, pancreas,parathyroid, peripheral nerve, pituitary, placenta, prostate, salivarygland, skin, small intestine, spinal cord, spleen, stomach, striatedmuscle, testis, thymus, thyroid, tonsil, ureter, urinary bladder, anduterus) from 3 unrelated adult monkeys obtained at autopsy or biopsy)are fixed and dried on object glass. The peroxidase staining of tissuesections is performed, using the avidin-biotin system.

The antibody or DVD-Ig molecule is incubated with the secondarybiotinylated anti-human IgG and developed into immune complex. Theimmune complex at the final concentrations of 2 and 10 μg/mL of antibodyor DVD-Ig molecule is added onto tissue sections on object glass andthen the tissue sections are reacted for 30 minutes with aavidin-biotin-peroxidase kit. Subsequently, DAB (3,3′-diaminobenzidine),a substrate for the peroxidase reaction, is applied for 4 minutes fortissue staining. Antigen-Sepharose beads are used as positive controltissue sections. Target antigen and human serum blocking studies serveas additional controls. The immune complex at the final concentrationsof 2 and 10 μg/mL of antibody or DVD-Ig molecule is pre-incubated withtarget antigen (final concentration of 100 g/ml) or human serum (finalconcentration 10%) for 30 minutes, and then added onto the tissuesections on object glass and then the tissue sections are reacted for 30minutes with a avidin-biotin-peroxidase kit. Subsequently, DAB(3,3′-diaminobenzidine), a substrate for the peroxidase reaction, isapplied for 4 minutes for tissue staining.

Any specific staining is judged to be either an expected (e.g.,consistent with antigen expression) or unexpected reactivity based uponknown expression of the target antigen in question. Any staining judgedspecific is scored for intensity and frequency. The tissue stainingbetween stage 2 (human tissue) and stage 3 (cynomolgus monkey tissue) iseither judged to be similar or different.

Example 1.1.2.E IL-1α/β Bioassay and Neutralization Assay Example1.1.2.E.1 Human IL-1α and IL-1β Bioassay for Monoclonal Antibodies

To examine the functional activity of the anti-human IL-1 alpha andanti-human IL-1 beta antibodies in the invention, the antibodies arescreened in a MRC-5 bioassay. The MRC-5 cell line is a human lungfibroblast cell line that produces human IL-8 in response to human IL-1alpha or beta in a dose-dependent manner. The MRC-5 cell line alsocross-reacts with cynomolgus IL-1 alpha and IL-1 beta. Antibody potencyis based on the ability of the antibody to inhibit IL-1 alpha or betainduced hIL-8 cytokine. MRC-5 cells (originally obtained from ATCC) aregrown and cultured in complete MEM containing 10% FBS in a 37° C., 5%CO₂ incubator. On the day prior to the assay, MRC5 cells are plated in a100 μL volume in a 96 well flat bottom plate (Costar #3599) at 1×10⁴then incubated overnight at 37° C., 5% CO₂. On the assay day, a working4× antibody and human IL-1α or human IL-1β, or cyno IL-1α or cyno IL-1βantigen stock is prepared in complete MEM medium. An eight point serialantibody dilution (10-0.0001 nM range) is performed in complete MEM inblock assay plates. Sixty-five μL of diluted antibody is transferred inquadruplicate to a 96 well v-bottom plate (Costar #3894) then 65 μL of a4× stock of appropriate antigen (200 pg/mL) is added to wells containingthe antibody. Sixty-five μL of antigen (200 pg/mL) is placed in antigencontrol wells with 65 μL of MEM media. Media control wells receive 130μL of MEM media. Following a 1 hour incubation, 100 μL of the Ab/Agmixture is added to the MRC-5 cells. Well volumes equaled 200 μL/welland all reagents are at a 1× final concentration. After an overnightincubation (16-24 hour), 150 μL of supernatant is transferred to a96-well round bottom plate (Costar #3799) and the plates are placed in a−20° C. freezer. The supernatants are tested for hIL-8 levels using ahuman IL-8 ELISA kit (R&D Systems, Minneapolis, Minn.) or a MSD hIL-8(chemiluminescent kit). Antibody neutralization potency is determined bycalculating percent inhibition relative to the antigen alone controlvalue.

Example 1.1.2.E.2 IL-1α/β Bioassay for Anti-IL-1α/β DVD-Ig Proteins

To examine the functional activity of anti-human IL-1 alpha and betaDVD-Ig proteins in the invention, the DVD-Ig proteins were screened in aMRC-5 bioassay. The MRC-5 cell line is a human lung fibroblast cell linethat produces human IL-8 in response to both human IL-1 alpha and betaand cyno IL-1 alpha and beta in a dose-dependent manner. DVD-Ig moleculepotency is based on the ability of the DVD-Ig molecule to inhibit IL-1induced hIL-8 cytokine. MRC-5 cells (originally obtained from ATCC) weregrown and cultured in complete MEM containing 10% FBS in a 37° C., 5%CO₂ incubator. On the day prior to the assay, MRC5 cells were plated ina 100 μL volume in a 96 well flat bottom plate (Costar #3599) at 1×10⁴then incubated overnight at 37° C., 5% CO₂. On the assay day, a 4×working stock of DVD-Ig molecule, hIL-1α and hIL-1β or cyno IL-1α andcyno IL-1β antigen stocks were prepared in complete MEM medium. On theassay day, an eight point DVD-Ig molecule serial dilution (100-0.0001 nMrange) was performed in complete MEM in block assay plates. Sixty-fiveμL of diluted DVD-Ig molecule was transferred in quadruplicate to a 96well v-bottom plate (Costar #3894) then 65 μL of either a 4× stock (200pg/mL) of IL-1α or IL-1β was added to wells containing DVD-Ig molecule.Sixty-five μL of IL-1α (200 pg/mL) or 65 μL of IL-1β (200 pg/mL) wasplaced in antigen control wells with 65 μL of MEM media. Media controlwells received 130 μL of MEM media. Following a 1 hour incubation, 100μL of the DVD-Ig/Ag mixture was added to the MRC-5 cells. Well volumesequaled 200 μL/well and all reagents were at a 1× final concentration.After an overnight incubation (16-24 hour), 150 μL of supernatant wastransferred to a 96-well round bottom plate (Costar #3799) and theplates were placed in a −20° C. freezer. The supernatants were testedfor hIL-8 levels by using a human IL-8 ELISA kit (R&D Systems,Minneapolis, Minn.) or MSD hIL-8 (chemiluminescence kit). DVD-Igmolecule neutralization potency was determined by calculating percentinhibition relative to the IL-1α or the IL-1β alone control value. Table7 demonstrates neutralization of human IL-1α by the IL-1α parentantibody and IL-1α and IL-1β DVD-Ig proteins. Table 8 demonstratesneutralization of human IL-1β by the IL-1β parent antibody and IL-1α andIL-1β DVD-Ig proteins. Table 9 demonstrates neutralization of cynomolgusIL-1α by the IL-1α parent antibody and IL-1α and IL-1β DVD-Ig proteins.Table 10 demonstrates neutralization of cynomolgus IL-1β by the IL-1βparent antibody and IL-1α and IL-1β DVD-Ig proteins.

TABLE 7 Human IL-1α Neutralization Assay With IL-1α Parent Antibody andDVD-Ig Constructs N- C- terminal terminal N-terminal VD C-terminal VDVariable Variable Human IL-1α Human IL-1α Parent Antibody or DomainDomain NeutralizationAssay NeutralizationAssay DVD-Ig ID (VD) (VD) EC50nM EC50 nM 3D12.10/15 IL-1α 0.0057 3D12r16 IL-1α 0.00056E26.35-SS-3D12r16 IL-1β IL-1α NA 0.0355 E26.13-SS- IL-1β IL-1α NA 0.05383D12.10/15 E26.35-SS- IL-1β IL-1α NA 0.0479 3D12.10/15 1B12.1-SS-3D12r16IL-1β IL-1α NA 0.0307 1B12.3-SS-3D12r16 IL-1β IL-1α NA 0.06871B12.6-SS-3D12r16 IL-1β IL-1α NA 0.0648 NA: Not applicable.

All DVD-Ig molecules showed neutralization in the MRC5 IL-1α/βneutralization assay.

TABLE 8 Human IL-1β Neutralization Assay With IL-1β Parent Antibody andDVD-Ig Constructs N- C- terminal terminal N-terminal VD C-terminal VDHuman Variable Variable Human IL-1β IL-1β Parent Antibody or DomainDomain NeutralizationAssay NeutralizationAssayEC50 DVD-Ig ID (VD) (VD)EC50 nM nM E26.13 IL-1β 0.0157 E26.13-SS- IL-1β IL-1α 0.0099 NA3D12.10/15 E26.35 IL-1β 0.0072 E26.35-SS-3D12.10/15 IL-1β IL-1α 0.002 NAE26.35-SS-3D12r16 IL-1β IL-1α 0.0029 NA 1B12.1 IL-1β 0.151B12.1-SS-3D12r16 IL-1β IL-1α 2.05 NA 1B12.3 IL-1β 0.301B12.3-SS-3D12r16 IL-1β IL-1α 1.74 NA 1B12.6 IL-1β 0.381B12.6-SS-3D12r16 IL-1β IL-1α 2.58 NA NA: Not applicable.

All DVD-Ig molecules showed neutralization in the MRC5 IL-1α/βneutralization assay.

TABLE 9 Cynomolgus IL-1α Neutralization Assay With IL-1α Parent Antibodyand DVD-Ig Constructs N- C- terminal terminal N-terminal VD C-terminalVD Variable Variable Cyno IL-1α Cyno IL-1α Parent Antibody or DomainDomain NeutralizationAssay NeutralizationAssay DVD-Ig ID (VD) (VD) EC50nM EC50 nM 3D12.10/15 IL-1α 0.0126 E26.13-SS-3D12.10/15 IL-1β IL-1α NA0.0626 E26.35-SS-3D12.10/15 IL-1β IL-1α NA 0.081 3D12r16 IL-1α 0.0008E26.35-SS-3D12r16 IL-1β IL-1α NA 0.1011 NA: Not applicable.

All DVD-Ig molecules showed neutralization in the MRC5 IL-1α/βneutralization assay.

TABLE 10 Cynomolgus IL-1β Neutralization Assay With IL-1β ParentAntibody and DVD-Ig Constructs N- C- terminal terminal N-terminal VDC-terminal VD Variable Variable Cyno IL-1β Cyno IL-1β Parent Antibody orDomain Domain NeutralizationAssay NeutralizationAssay DVD-Ig ID (VD)(VD) EC50 nM EC50 nM E26.13 IL-1β 0.0084 E26.13-SS-3D12.10/15 IL-1βIL-1α 0.0056 NA E26.35 IL-1β 0.0034 E26.35-SS-3D12.10/15 IL-1β IL-1α0.0121 NA E26.35-SS-3D12r16 IL-1β IL-1α 0.004 NA NA: Not applicable.

All DVD-Ig molecules showed neutralization in the MRC5 IL-1α/βneutralization assay.

Example 1.1.2.F Growth Inhibitory Effect of a Tumor Receptor MonoclonalAntibody or DVD-Ig Molecules In Vitro

Tumor receptor monoclonal antibodies or DVD-Ig molecules diluted inD-PBS-BSA (Dulbecco's phosphate buffered saline with 0.1% BSA) 20 μL areadded to human tumor cells at final concentrations of 0.01 μg/mL-100μg/mL in 180 μL. The plates are incubated at 37° C. in a humidified, 5%CO₂ atmosphere for 3 days. The number of live cells in each well isquantified using MTS reagents according to the manufacturer'sinstructions (Promega, Madison, Wis.) to determine the percent of tumorgrowth inhibition. Wells without antibody treatment are used as controlsof 0% inhibition whereas wells without cells are considered to show 100%inhibition.

Example 1.1.2.G Tumoricidal Effect of A Parent Antibody or DVD-IgMolecule In Vitro

Parent antibodies or DVD-Ig molecules that bind to target antigens ontumor cells may be analyzed for tumoricidal activity. Briefly, parentantibodies or DVD-Ig molecule are diluted in D-PBS-BSA (Dulbecco'sphosphate buffered saline with 0.1% BSA) and added to human tumor cellsat final concentrations of 0.01 μg/mL to 100 μg/mL in 200 μL. The platesare incubated at 37° C. in a humidified, 5% CO₂ atmosphere for 3 days.The number of live cells in each well is quantified using MTS reagentsaccording to the manufacturer's instructions (Promega, Madison, Wis.) todetermine the percent of tumor growth inhibition. Wells without antibodytreatment are used as controls of 0% inhibition whereas wells withoutcells were considered to show 100% inhibition.

For assessment of apoptosis, caspase-3 activation is determined by thefollowing protocol: antibody-treated cells in 96 well plates are lysedin 120 μl of 1× lysis buffer (1.67 mM Hepes, pH 7.4, 7 mM KCl, 0.83 mMMgCl₂, 0.11 mM EDTA, 0.11 mM EGTA, 0.57% CHAPS, 1 mM DTT, 1× proteaseinhibitor cocktail tablet; EDTA-free; Roche Pharmaceuticals, Nutley,N.J.) at room temperature with shaking for 20 minutes. After cell lysis,80 μl of a caspase-3 reaction buffer (48 mM Hepes, pH 7.5, 252 mMsucrose, 0.1% CHAPS, 4 mM DTT, and 20 μM Ac-DEVD (SEQ ID NO: 78)-AMCsubstrate; Biomol Research Labs, Inc., Plymouth Meeting, Pa.) is addedand the plates are incubated for 2 hours at 37° C. The plates are readon a 1420 VICTOR Multilabel Counter (Perkin Elmer Life Sciences, DownersGrove, Ill.) using the following settings: excitation=360/40,emission=460/40. An increase of fluorescence units from antibody-treatedcells relative to the isotype antibody control-treated cells isindicative of apoptosis.

Example 1.1.2.H Inhibition of Cell Proliferation by Parent Antibody andDVD-Ig Constructs

U87-MG human glioma tumor cells are plated at 2,000 cells/well in 100 μlin 96-well dishes in RPMI medium supplemented with 5% fetal bovineserum, and incubated at 37° C., 5% CO₂ overnight. The following day thecells are treated with serial dilutions of antibody or DVD-Ig molecules(0.013 nM to 133 nM dose range), and incubated at 37° C. in ahumidified, 5% CO₂ atmosphere for 5 days. Cell survival/proliferation ismeasured indirectly by assessing ATP levels using an ATPlite kit (PerkinElmer, Waltham, Mass.) according to the manufacturer's instructions.

Example 1.1.2.1 Inhibition of Receptor Phosphorylation by ParentAntibodies or DVD-Ig Constructs In Vitro

Human carcinoma cells are plated in 96-well plates at 40,000 cells/wellin 180 μl serum-free medium (DMEM+0.1% BSA), and incubated overnight at37° C., 5% CO₂. Costar EIA plates (Lowell, Mass.) are coated with 100μl/well of receptor capture Ab (4 μg/ml final concentration), andincubated overnight at room temperature while shaking. The followingday, receptor antibody-coated ELISA plates are washed (three times withPBST=0.05% Tween 20 in PBS, pH 7.2-7.4), and 200 μl blocking solution isadded (1% BSA, 0.05% NaN3 in PBS, pH 7.2-7.4) to block for 2 hours atroom temperature on a rocker. Human tumor cells are co-incubated withantibodies or DVD-Ig molecules and ligand. Monoclonal antibodies orDVD-Ig molecules diluted in D-PBS-BSA (Dulbecco's phosphate bufferedsaline with 0.1% BSA) are added to human carcinoma cells at finalconcentrations of 0.01 μg/mL-100 μg/mL. Growth factors aresimultaneously added to the cells at concentrations of 1-100 ng/mL (200μL), and cells are incubated at 37° C. in a humidified, 5% CO₂atmosphere for 1 hour. Cells are lysed in 120 μl/well of cold cellextraction buffer (10 mM Tris, pH 7.4, 100 mM NaCl, 1 mM EDTA, 1 mMEGTA, 1 mM NaF, 1 mM sodium orthovanadate, 1% Triton X-100, 10%Glycerol, 0.1% SDS, and protease inhibitor cocktail), and incubated at4° C. for 20 minutes with shaking. Cell lysates (100 μl) are added tothe ELISA plate, and incubated overnight at 4° C. with gentle shaking.The following day, ELISA plates are washed, and 100 μl/well of pTyr-HRPdetection Ab is added (p-IGF1R ELISA kit, R&D System #DYC 1770,Minneapolis, Minn.), and plates are incubated for 2 hours at 25° C. inthe dark. Plates are developed to determine phosphorylation per themanufacturer's instructions.

Example 1.1.2.J Efficacy of a DVD-Ig Molecule on the Growth of HumanCarcinoma Subcutaneous Flank Xenografts

A-431 human epidermoid carcinoma cells are grown in vitro to 99%viability, 85% confluence in tissue culture flasks. SCID female mice(Charles Rivers Labs, Wilmington, Mass.) at 19-25 grams are injectedsubcutaneously into the right flank with 1×10⁶ human tumor cells (1:1matrigel) on study day 0. Administration (IP, QD, 3×/week) of human IgGcontrol or DVD-Ig molecule was-initiated after mice are size matchedinto groups of mice with mean tumor volumes of approximately 200 to 320mm³. The tumors are measured twice a week starting on approximately day10 post tumor cell injection.

Example 1.2 Generation of Parent Monoclonal Antibodies to a HumanAntigen of Interest

Parent mouse mAbs that bind to and neutralize a human antigen ofinterest and a variant thereof are obtained as follows:

Example 1.2.A Immunization of Mice with a Human Antigen of Interest

Twenty micrograms of recombinant purified human antigen (e.g., IL-1α orIL-1β) mixed with complete Freund's adjuvant or Immunoeasy adjuvant(Qiagen, Valencia, Calif.) is injected subcutaneously into five 6-8week-old Balb/C, five C57B/6 mice, and five AJ mice on Day 1. On days24, 38, and 49, twenty micrograms of recombinant purified human antigenvariant mixed with incomplete Freund's adjuvant or Immunoeasy adjuvantis injected subcutaneously into the same mice. On day 84 or day 112 orday 144, mice are injected intravenously with 1 μg recombinant purifiedhuman antigen of interest.

Example 1.2.B Generation of a Hybridoma

Splenocytes obtained from the immunized mice described in Example 1.2.Aare fused with SP2/O-Ag-14 cells at a ratio of 5:1 according to theestablished method described in Kohler, G. and Milstein (1975) Nature,256:495 to generate hybridomas. Fusion products are plated in selectionmedia containing azaserine and hypoxanthine in 96-well plates at adensity of 2.5×10⁶ spleen cells per well. Seven to ten days post fusion,macroscopic hybridoma colonies are observed. Supernatant from each wellcontaining hybridoma colonies is tested by ELISA for the presence ofantibody to the antigen of interest (e.g., as described in Example1.1.1.A). Supernatants displaying antigen-specific activity are thentested for activity (e.g., as described in the assays of Example 1.1.2),for example, the ability to neutralize the antigen of interest in abioassay, e.g., such as that described in Example 1.1.2.E).

Example 1.2.C Identification and Characterization of Parent MonoclonalAntibodies to a Human Target Antigen of Interest Example 1.2.C.1Analyzing Parent Monoclonal Antibody Neutralizing Activity

Hybridoma supernatants are assayed for the presence of parent antibodiesthat bind an antigen of interest, generated according to Examples 1.2.Aand 1.2.B, and are also capable of binding a variant of the antigen ofinterest (“antigen variant”). Supernatants with antibodies positive inboth assays are then tested for their antigen neutralization potency,for example, in a cytokine bioassay of Example 1.1.2.E. The hybridomasproducing antibodies with IC₅₀ values in the bioassay less than 1000 pM,in an embodiment, less than 100 pM are scaled up and cloned by limitingdilution. Hybridoma cells are expanded into media containing 10% low IgGfetal bovine serum (Hyclone #SH30151, Logan, Utah). On average, 250 mLof each hybridoma supernatant (derived from a clonal population) isharvested, concentrated and purified by protein A affinitychromatography, as described in Harlow and Lane (1988) Antibodies: ALaboratory Manual. The ability of purified mAbs to inhibit the activityof its target antigen is determined, for example, using a cytokinebioassay as described in Example 1.1.2.E.

Example 1.2.C.2 Analyzing Parent Monoclonal Antibody Cross-Reactivity toCynomolgus Target Antigen of Interest

To determine whether the selected mAbs described herein recognizecynomolgus antigen of interest, BIACORE analysis is conducted asdescribed herein (Example 1.1.1.C) using recombinant cynomolgus targetantigen. In addition, neutralization potencies of mAbs againstrecombinant cynomolgus antigen of interest may also be measured in acytokine bioassay (Example 1.1.2.E). MAbs with good cynocross-reactivity (in an embodiment, within 5-fold of reactivity forhuman antigen) are selected for future characterization.

Example 1.2.D Determination of the Amino Acid Sequence of the VariableRegion for Each Murine Anti-Human Monoclonal Antibody

Isolation of the cDNAs, expression and characterization of therecombinant anti-human mouse mAbs is conducted as follows. For eachamino acid sequence determination, approximately 1×10⁶ hybridoma cellsare isolated by centrifugation and processed to isolate total RNA withTrizol (Gibco BRL/Invitrogen, Carlsbad, Calif.) following manufacturer'sinstructions. Total RNA is subjected to first strand DNA synthesis usingthe SuperScript First-Strand Synthesis System (Invitrogen, Carlsbad,Calif.) per the manufacturer's instructions. Oligo(dT) is used to primefirst-strand synthesis to select for poly(A)+ RNA. The first-strand cDNAproduct is then amplified by PCR with primers designed for amplificationof murine immunoglobulin variable regions (Ig-Primer Sets, Novagen,Madison, Wis.). PCR products are resolved on an agarose gel, excised,purified, and then subcloned with the TOPO Cloning kit into pCR2.1-TOPOvector (Invitrogen, Carlsbad, Calif.) and transformed into TOP10chemically competent E. coli (Invitrogen, Carlsbad, Calif.). Colony PCRis performed on the transformants to identify clones containing insert.Plasmid DNA is isolated from clones containing insert using a QIAprepMiniprep kit (Qiagen, Valencia, Calif.). Inserts in the plasmids aresequenced on both strands to determine the variable heavy or variablelight chain DNA sequences using M13 forward and M13 reverse primers(Fermentas Life Sciences, Hanover Md.). Variable heavy and variablelight chain sequences of the mAbs are identified. In an embodiment, theselection criteria for a panel of lead mAbs for next step development(humanization) includes the following:

-   -   The antibody does not contain any N-linked glycosylation sites        (NXS), except from the standard one in CH2    -   The antibody does not contain any extra cysteines in addition to        the normal cysteines in every antibody    -   The antibody sequence is aligned with the closest human germline        sequences for VH and VL and any unusual amino acids should be        checked for occurrence in other natural human antibodies    -   N-terminal Glutamine (Q) is changed to Glutamic acid (E) if it        does not affect the activity of the antibody. This will reduce        heterogeneity due to cyclization of Q    -   Efficient signal sequence cleavage is confirmed by Mass        Spectrophotometry. This can be done with COS cell or 293 cell        material    -   The protein sequence is checked for the risk of deamidation of        Asn that could result in loss of activity    -   The antibody has a low level of aggregation    -   The antibody has solubility >5-10 mg/ml (in research phase); >25        mg/ml    -   The antibody has a normal size (5-6 nm) by Dynamic Light        Scattering (DLS)    -   The antibody has a low charge heterogeneity    -   The antibody lacks cytokine release (see Example 1.1.2.B)    -   The antibody has specificity for the intended cytokine (see        Example 1.1.2.C)    -   The antibody lacks unexpected tissue cross reactivity (see        Example 1.1.2.D)    -   The antibody has similarity between human and cynomolgus tissue        cross reactivity (see Example 1.1.2.D)

Example 1.2.2 Recombinant Humanized Parent Antibodies Example 1.2.2.1Construction and Expression of Recombinant Chimeric Anti Human ParentAntibodies

The DNA encoding the heavy chain constant region of murine anti-humanparent mAbs is replaced by a cDNA fragment encoding the human IgG1constant region containing 2 hinge-region amino acid mutations byhomologous recombination in bacteria. These mutations are a leucine toalanine change at position 234 (EU numbering) and a leucine to alaninechange at position 235 (Lund et al. (1991) J. Immunol. 147:2657). Thelight chain constant region of each of these antibodies is replaced by ahuman kappa constant region. Full-length chimeric antibodies aretransiently expressed in COS cells by co-transfection of chimeric heavyand light chain cDNAs ligated into the pBOS expression plasmid(Mizushima and Nagata (1990) Nucl. Acids Res. 18: 5322). Cellsupernatants containing recombinant chimeric antibody are purified byProtein A Sepharose chromatography and bound antibody is eluted byaddition of acid buffer. Antibodies are neutralized and dialyzed intoPBS.

The heavy chain cDNA encoding a chimeric mAb is co-transfected with itschimeric light chain cDNA (both ligated in the pBOS vector) into COScells. Cell supernatant containing recombinant chimeric antibody ispurified by Protein A Sepharose chromatography and bound antibody iseluted by addition of acid buffer. Antibodies are neutralized anddialyzed into PBS.

The purified chimeric anti-human parent mAbs are then tested for theirability to bind (by Biacore) and for functional activity, e.g., to asdescribed in Examples 1.1.1.C and 1.1.2.B. Chimeric mAbs that maintainthe activity of the parent hybridoma mAbs are selected for futuredevelopment.

Example 1.2.2.2 Construction and Expression of Humanized Anti HumanParent Antibodies Example 1.2.2.2.A Selection of Human AntibodyFrameworks

Each murine variable heavy and variable light chain gene sequence isseparately aligned against 44 human immunoglobulin germline variableheavy chain or 46 germline variable light chain sequences (derived fromNCBI Ig Blast website at www.ncbi.nlm.nih gov/igblast/retrieveig.html.)using Vector NTI software.

Humanization is based on amino acid sequence homology, CDR clusteranalysis, frequency of use among expressed human antibodies, andavailable information on the crystal structures of human antibodies.Taking into account possible effects on antibody binding, VH-VL pairing,and other factors, murine residues are mutated to human residues wheremurine and human framework residues are different, with a fewexceptions. Additional humanization strategies are designed based on ananalysis of human germline antibody sequences, or a subgroup thereof,that possessed a high degree of homology, i.e., sequence similarity, tothe actual amino acid sequence of the murine antibody variable regions.

Homology modeling is used to identify residues unique to the murineantibody sequences that are predicted to be critical to the structure ofthe antibody combining site, the CDRs. Homology modeling is acomputational method whereby approximate three dimensional coordinatesare generated for a protein. The source of initial coordinates andguidance for their further refinement is a second protein, the referenceprotein, for which the three dimensional coordinates are known and thesequence of which is related to the sequence of the first protein. Therelationship among the sequences of the two proteins is used to generatea correspondence between the reference protein and the protein for whichcoordinates are desired, the target protein. The primary sequences ofthe reference and target proteins are aligned with coordinates ofidentical portions of the two proteins transferred directly from thereference protein to the target protein. Coordinates for mismatchedportions of the two proteins, e.g., from residue mutations, insertions,or deletions, are constructed from generic structural templates andenergy refined to insure consistency with the already transferred modelcoordinates. This computational protein structure may be further refinedor employed directly in modeling studies. The quality of the modelstructure is determined by the accuracy of the contention that thereference and target proteins are related and the precision with whichthe sequence alignment is constructed.

For the murine mAbs, a combination of BLAST searching and visualinspection is used to identify suitable reference structures. Sequenceidentity of 25% between the reference and target amino acid sequences isconsidered the minimum necessary to attempt a homology modelingexercise. Sequence alignments are constructed manually and modelcoordinates are generated with the program Jackal (see Petrey et al.(2003) Proteins 53 (Suppl. 6): 430-435).

The primary sequences of the murine and human framework regions of theselected antibodies share significant identity. Residue positions thatdiffer are candidates for inclusion of the murine residue in thehumanized sequence in order to retain the observed binding potency ofthe murine antibody. A list of framework residues that differ betweenthe human and murine sequences is constructed manually. Table 11 showsthe framework sequences chosen for this study.

TABLE 11 Sequence Of Human IgG Heavy Chain Constant Domain AndLight Chain Constant Domain SEQ Sequence Protein ID NO1234567890123456789012345678901234567890 Wild type hIgG1 48ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVS constant regionWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPE LL GGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYT QKSLSLSPGK Mutant hIgG1 49ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVS constant regionWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPE AA GGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYT QKSLSLSPGK Ig Kappa 50TVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQW constant regionKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEK HKVYACEVTHQGLSSPVTKSFNRGECIg Lambda 51 QPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVA constant regionWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKS HRSYSCQVTHEGSTVEKTVAPTECS

The likelihood that a given framework residue would impact the bindingproperties of the antibody depends on its proximity to the CDR residues.Therefore, using the model structures, the residues that differ betweenthe murine and human sequences are ranked according to their distancefrom any atom in the CDRs. Those residues that fell within 4.5 Å of anyCDR atom are identified as most important and are recommended to becandidates for retention of the murine residue in the humanized antibody(i.e., back mutation).

In silico constructed humanized antibodies are constructed usingoligonucleotides. For each variable region cDNA, 6 oligonucleotides of60-80 nucleotides each are designed to overlap each other by 20nucleotides at the 5′ and/or 3′ end of each oligonucleotide. In anannealing reaction, all 6 oligonucleotides are combined, boiled, andannealed in the presence of dNTPs. DNA polymerase I, Large (Klenow)fragment (New England Biolabs #M0210, Beverley, Mass.) is added tofill-in the approximately 40 bp gaps between the overlappingoligonucleotides. PCR is performed to amplify the entire variable regiongene using two outermost primers containing overhanging sequencescomplementary to the multiple cloning site in a modified pBOS vector(Mizushima and Nagata (1990) Nucl. Acids Res. 18: 17). The PCR productsderived from each cDNA assembly are separated on an agarose gel and theband corresponding to the predicted variable region cDNA size is excisedand purified. The variable heavy region is inserted in-frame onto a cDNAfragment encoding the human IgG1 constant region containing 2hinge-region amino acid mutations by homologous recombination inbacteria. These mutations are a leucine to alanine change at position234 (EU numbering) and a leucine to alanine change at position 235 (Lundet al. (1991) J. Immunol. 147:2657). The variable light chain region isinserted in-frame with the human kappa constant region by homologousrecombination. Bacterial colonies are isolated and plasmid DNAextracted. cDNA inserts are sequenced in their entirety. Correcthumanized heavy and light chains corresponding to each antibody areco-transfected into COS cells to transiently produce full-lengthhumanized anti-human antibodies. Cell supernatants containingrecombinant chimeric antibody are purified by Protein A Sepharosechromatography and bound antibody is eluted by addition of acid buffer.Antibodies are neutralized and dialyzed into PBS.

Example 1.2.2.3 Characterization of Humanized Antibodies

The ability of purified humanized antibodies to inhibit a functionalactivity is determined, e.g., using the cytokine bioassay as describedin Examples 1.1.2.A. The binding affinities of the humanized antibodiesto recombinant human antigen are determined using surface plasmonresonance (Biacore®) measurement as described in Example 1.1.1.B. TheIC₅₀ values from the bioassays and the affinity of the humanizedantibodies are ranked. The humanized mAbs that fully maintain theactivity of the parent hybridoma mAbs are selected as candidates forfuture development. The top 2-3 most favorable humanized mAbs arefurther characterized.

Example 1.2.2.3.A Pharmacokinetic Analysis of Humanized Antibodies

Pharmacokinetic studies are carried out in Sprague-Dawley rats andcynomolgus monkeys. Male and female rats and cynomolgus monkeys aredosed intravenously or subcutaneously with a single dose of 4 mg/kg mAband samples are analyzed using antigen capture ELISA, andpharmacokinetic parameters are determined by noncompartmental analysis.Briefly, ELISA plates are coated with goat anti-biotin antibody (5mg/ml, 4° C., overnight), blocked with Superblock (Pierce), andincubated with biotinylated human antigen at 50 ng/ml in 10% SuperblockTTBS at room temperature for 2 hours. Serum samples are serially diluted(0.5% serum, 10% Superblock in TTBS) and incubated on the plate for 30minutes at room temperature. Detection is carried out with HRP-labeledgoat anti human antibody and concentrations are determined with the helpof standard curves using the four parameter logistic fit. Values for thepharmacokinetic parameters are determined by non-compartmental modelusing WinNonlin software (Pharsight Corporation, Mountain View, Calif.).Humanized mAbs with good pharmacokinetics profile (T½ is 8-13 days orbetter, with low clearance and excellent bioavailability 50-100%) areselected.

Example 1.2.2.3.B Physicochemical and In Vitro Stability Analysis ofHumanized Monoclonal Antibodies

Size Exclusion Chromatography

Antibodies are diluted to 2.5 mg/mL with water and 20 mL is analyzed ona Shimadzu HPLC system using a TSK gel G3000 SWXL column (TosohBioscience, cat #k5539-05k). Samples are eluted from the column with 211mM sodium sulfate, 92 mM sodium phosphate, pH 7.0, at a flow rate of 0.3mL/minutes. The HPLC system operating conditions are the following:

Mobile phase: 211 mM Na₂SO₄, 92 mM Na₂HPO₄*7H₂O, pH 7.0

Gradient: Isocratic

Flow rate: 0.3 mL/minute

Detector wavelength: 280 nm

Autosampler cooler temp: 4° C.

Column oven temperature: Ambient

Run time: 50 minutes

Table 12 contains purity data of parent antibodies and DVD-Ig constructsexpressed as percent monomer (unaggregated protein of the expectedmolecular weight) as determined by the above protocol.

TABLE 12 Purity of Parent Antibodies and DVD-Ig Constructs as Determinedby Size Exclusion Chromatography N-Terminal C-Terminal Variable VariableParent Antibody Domain Domain or DVD-Ig ID (VD) (VD) % Monomer (purity)E26.13 IL-1β 99.0 3D12.10/15 IL-1α 81.4 E26.13-SS- IL-1β IL-1α 95.13D12.10/15 E26.35 IL-1β 88.1 3D12r16 IL-1α 100 E26.35-SS- IL-1β IL-1α97.4 3D12.10/15 E26.35-SS- IL-1β IL-1α 100 3D12r16 1B12.1-SS- IL-1βIL-1α 95.1 3D12r16 1B12.3-SS- IL-1β IL-1α 94.6 3D12r16 1B12.6-SS- IL-1βIL-1α 95.9 3D12r16

DVD-Ig molecules showed an excellent SEC profile with most DVD-Igmolecule showing >94% monomer. This DVD-Ig profile is similar to thatobserved for parent antibodies.

SDS-PAGE

Antibodies are analyzed by sodium dodecyl sulfate-polyacrylamide gelelectrophoresis (SDS-PAGE) under both reducing and non-reducingconditions. Adalimumab lot AFP04C is used as a control. For reducingconditions, the samples are mixed 1:1 with 2× tris glycine SDS-PAGEsample buffer (Invitrogen, cat #LC2676, lot #1323208) with 100 mM DTT,and heated at 60° C. for 30 minutes. For non-reducing conditions, thesamples are mixed 1:1 with sample buffer and heated at 100° C. for 5minutes. The reduced samples (10 mg per lane) are loaded on a 12%pre-cast tris-glycine gel (Invitrogen, cat #EC6005box, lot #6111021),and the non-reduced samples (10 mg per lane) are loaded on an 8%-16%pre-cast tris-glycine gel (Invitrogen, cat #EC6045box, lot #6111021).SeeBlue Plus 2 (Invitrogen, cat #LC5925, lot #1351542) is used as amolecular weight marker. The gels are run in a XCell SureLock mini cellgel box (Invitrogen, cat #E10001) and the proteins are separated byfirst applying a voltage of 75 to stack the samples in the gel, followedby a constant voltage of 125 until the dye front reached the bottom ofthe gel. The running buffer used is lx tris glycine SDS buffer, preparedfrom a 10× tris glycine SDS buffer (ABC, MPS-79-080106)). The gels arestained overnight with colloidal blue stain (Invitrogen cat #46-7015,46-7016) and destained with Milli-Q water until the background is clear.The stained gels are then scanned using an Epson Expression scanner(model 1680, S/N DASX003641).

Sedimentation Velocity Analysis

Antibodies are loaded into the sample chamber of each of three standardtwo-sector carbon epon centerpieces. These centerpieces have a 1.2 cmoptical path length and are built with sapphire windows. PBS is used fora reference buffer and each chamber contained 140 μL. All samples areexamined simultaneously using a 4-hole (AN-60Ti) rotor in a BeckmanProteomeLab XL-I analytical ultracentrifuge (serial #PL106C01).

Run conditions are programmed and centrifuge control is performed usingProteomeLab (v5.6). The samples and rotor are allowed to thermallyequilibrate for one hour prior to analysis (20.0±0.1° C.). Confirmationof proper cell loading is performed at 3000 rpm and a single scan isrecorded for each cell. The sedimentation velocity conditions are thefollowing:

Sample Cell Volume: 420 mL

Reference Cell Volume: 420 mL

Temperature: 20° C.

Rotor Speed: 35,000 rpm

Time: 8:00 hours

UV Wavelength: 280 nm

Radial Step Size: 0.003 cm

Data Collection: One data point per step without signal averaging.

Total Number of Scans: 100

LC-MS Molecular Weight Measurement of Intact Antibodies

Molecular weight of intact antibodies are analyzed by LC-MS. Eachantibody is diluted to approximately 1 mg/mL with water. An 1100 HPLC(Agilent) system with a protein microtrap (Michrom Bioresources, Inc,cat #004/25109/03) is used to desalt and introduce 5 mg of the sampleinto an API Qstar pulsar i mass spectrometer (Applied Biosystems). Ashort gradient is used to elute the samples. The gradient is run withmobile phase A (0.08% FA, 0.02% TFA in HPLC water) and mobile phase B(0.08% FA and 0.02% TFA in acetonitrile) at a flow rate of 50 mL/minute.The mass spectrometer is operated at 4.5 kvolts spray voltage with ascan range from 2000 to 3500 mass to charge ratio.

LC-MS Molecular Weight Measurement of Antibody Light and Heavy Chains

Molecular weight measurement of antibody light chain (LC), heavy chain(HC) and deglycosylated HC are analyzed by LC-MS. Antibody is diluted to1 mg/mL with water and the sample is reduced to LC and HC with a finalconcentration of 10 mM DTT for 30 minutes at 37° C. To deglycosylate theantibody, 100 mg of the antibody is incubated with 2 mL of PNGase F, 5mL of 10% N-octylglucoside in a total volume of 100 mL overnight at 37°C. After deglycosylation the sample is reduced with a finalconcentration of 10 mM DTT for 30 minutes at 37° C. An Agilent 1100 HPLCsystem with a C4 column (Vydac, cat #214TP5115, S/N 060206537204069) isused to desalt and introduce the sample (5 mg) into an API Qstar pulsari mass spectrometer (Applied Biosystems). A short gradient is used toelute the sample. The gradient is run with mobile phase A (0.08% FA,0.02% TFA in HPLC water) and mobile phase B (0.08% FA and 0.02% TFA inacetonitrile) at a flow rate of 50 mL/minute. The mass spectrometer isoperated at 4.5 kvolts spray voltage with a scan range from 800 to 3500mass to charge ratio.

Peptide Mapping

Antibody is denatured for 15 minutes at room temperature with a finalconcentration of 6 M guanidine hydrochloride in 75 mM ammoniumbicarbonate. The denatured samples are reduced with a finalconcentration of 10 mM DTT at 37° C. for 60 minutes, followed byalkylation with 50 mM iodoacetic acid (IAA) in the dark at 37° C. for 30minutes. Following alkylation, the sample is dialyzed overnight againstfour liters of 10 mM ammonium bicarbonate at 4° C. The dialyzed sampleis diluted to 1 mg/mL with 10 mM ammonium bicarbonate, pH 7.8 and 100 mgof antibody is either digested with trypsin (Promega, cat #V5111) orLys-C (Roche, cat #11 047 825 001) at a 1:20 (w/w)trypsin/Lys-C:antibody ratio at 37° C. for 4 hrs. Digests are quenchedwith 1 mL of 1 N HCl. For peptide mapping with mass spectrometerdetection, 40 mL of the digests are separated by reverse phase highperformance liquid chromatography (RPHPLC) on a C18 column (Vydac, cat#218TP51, S/N NE9606 10.3.5) with an Agilent 1100 HPLC system. Thepeptide separation is run with a gradient using mobile phase A (0.02%TFA and 0.08% FA in HPLC grade water) and mobile phase B (0.02% TFA and0.08% FA in acetonitrile) at a flow rate of 50 mL/minutes. The API QSTARPulsar i mass spectromer is operated in positive mode at 4.5 kvoltsspray voltage and a scan range from 800 to 2500 mass to charge ratio.

Disulfide Bond Mapping

To denature the antibody, 100 mL of the antibody is mixed with 300 mL of8 M guanidine HCl in 100 mM ammonium bicarbonate. The pH is checked toensure that it is between 7 and 8 and the samples are denatured for 15minutes at room temperature in a final concentration of 6 M guanidineHCl. A portion of the denatured sample (100 mL) is diluted to 600 mLwith Milli-Q water to give a final guanidine-HCl concentration of 1 M.The sample (220 mg) is digested with either trypsin (Promega, cat#V5111, lot #22265901) or Lys-C (Roche, cat #11047825001, lot #12808000)at a 1:50 trypsin or 1:50 Lys-C: antibody (w/w) ratios (4.4 mg enzyme:220 mg sample) at 37° C. for approximately 16 hours. An additional 5 mgof trypsin or Lys-C is added to the samples and digestion is allowed toproceed for an additional 2 hours at 37° C. Digestions are stopped byadding 1 mL of TFA to each sample. Digested samples are separated byRPHPLC using a C18 column (Vydac, cat #218TP51 S/N NE020630-4-1A) on anAgilent HPLC system. The separation is run with the same gradient usedfor peptide mapping using mobile phase A (0.02% TFA and 0.08% FA in HPLCgrade water) and mobile phase B (0.02% TFA and 0.08% FA in acetonitrile)at a flow rate of 50 mL/minute. The HPLC operating conditions are thesame as those used for peptide mapping. The API QSTAR Pulsar i massspectromer is operated in positive mode at 4.5 kvolts spray voltage anda scan range from 800 to 2500 mass-to-charge ratio. Disulfide bonds areassigned by matching the observed MWs of peptides with the predicted MWsof tryptic or Lys-C peptides linked by disulfide bonds.

Free Sulfhydryl Determination

The method used to quantify free cysteines in an antibody is based onthe reaction of Ellman's reagent, 5,5′-dithio-bis(2-nitrobenzoic acid)(DTNB), with sulfhydryl groups (SH) which gives rise to a characteristicchromophoric product, 5-thio-(2-nitrobenzoic acid) (TNB). The reactionis illustrated in the formula:DTNB+RSH®RS−TNB+TNB−+H+

The absorbance of the TNB− is measured at 412 nm using a Cary 50spectrophotometer. An absorbance curve is plotted using dilutions of 2mercaptoethanol (b-ME) as the free SH standard and the concentrations ofthe free sulfhydryl groups in the protein are determined from absorbanceat 412 nm of the sample.

The b-ME standard stock is prepared by a serial dilution of 14.2 M b-MEwith HPLC grade water to a final concentration of 0.142 mM. Thenstandards in triplicate for each concentration are prepared. Antibody isconcentrated to 10 mg/mL using an amicon ultra 10,000 MWCO centrifugalfilter (Millipore, cat #UFC801096, lot #L3KN5251) and the buffer ischanged to the formulation buffer used for adalimumab (5.57 mM sodiumphosphate monobasic, 8.69 mM sodium phosphate dibasic, 106.69 mM NaCl,1.07 mM sodium citrate, 6.45 mM citric acid, 66.68 mM mannitol, pH 5.2,0.1% (w/v) Tween). The samples are mixed on a shaker at room temperaturefor 20 minutes. Then 180 mL of 100 mM Tris buffer, pH 8.1 is added toeach sample and standard followed by the addition of 300 mL of 2 mM DTNBin 10 mM phosphate buffer, pH 8.1. After thorough mixing, the samplesand standards are measured for absorption at 412 nm on a Cary 50spectrophotometer. The standard curve is obtained by plotting the amountof free SH and OD₄₁₂ nm of the b-ME standards. Free SH content ofsamples are calculated based on this curve after subtraction of theblank.

Weak Cation Exchange Chromatography

Antibody is diluted to 1 mg/mL with 10 mM sodium phosphate, pH 6.0.Charge heterogeneity is analyzed using a Shimadzu HPLC system with aWCX-10 ProPac analytical column (Dionex, cat #054993, S/N 02722). Thesamples are loaded on the column in 80% mobile phase A (10 mM sodiumphosphate, pH 6.0) and 20% mobile phase B (10 mM sodium phosphate, 500mM NaCl, pH 6.0) and eluted at a flow rate of 1.0 mL/minute.

Oligosaccharide Profiling

Oligosaccharides released after PNGase F treatment of antibody arederivatized with 2-aminobenzamide (2-AB) labeling reagent. Thefluorescent-labeled oligosaccharides are separated by normal phase highperformance liquid chromatography (NPHPLC) and the different forms ofoligosaccharides are characterized based on retention time comparisonwith known standards.

The antibody is first digested with PNGaseF to cleave N-linkedoligosaccharides from the Fc portion of the heavy chain. The antibody(200 mg) is placed in a 500 mL Eppendorf tube along with 2 mL PNGase Fand 3 mL of 10% N-octylglucoside. Phosphate buffered saline is added tobring the final volume to 60 mL. The sample is incubated overnight at37° C. in an Eppendorf thermomixer set at 700 RPM. Adalimumab lot AFP04Cis also digested with PNGase F as a control.

After PNGase F treatment, the samples are incubated at 95° C. for 5minutes in an Eppendorf thermomixer set at 750 RPM to precipitate outthe proteins, then the samples are placed in an Eppendorf centrifuge for2 minutes at 10,000 RPM to spin down the precipitated proteins. Thesupernatent containing the oligosaccharides are transferred to a 500 mLEppendorf tube and dried in a speed-vac at 65° C.

The oligosaccharides are labeled with 2AB using a 2AB labeling kitpurchased from Prozyme (cat #GKK-404, lot #132026). The labeling reagentis prepared according to the manufacturer's instructions. Acetic acid(150 mL, provided in kit) is added to the DMSO vial (provided in kit)and mixed by pipetting the solution up and down several times. Theacetic acid/DMSO mixture (100 mL) is transferred to a vial of 2-AB dye(just prior to use) and mixed until the dye is fully dissolved. The dyesolution is then added to a vial of reductant (provided in kit) andmixed well (labeling reagent). The labeling reagent (5 mL) is added toeach dried oligosaccharide sample vial, and mixed thoroughly. Thereaction vials are placed in an Eppendorf thermomixer set at 65° C. and700-800 RPM for 2 hours of reaction.

After the labeling reaction, the excess fluorescent dye is removed usingGlycoClean S Cartridges from Prozyme (cat #GKI-4726). Prior to addingthe samples, the cartridges are washed with 1 mL of milli-Q waterfollowed with 5 ishes of 1 mL 30% acetic acid solution. Just prior toadding the samples, 1 mL of acetonitrile (Burdick and Jackson, cat#AH015-4) is added to the cartridges.

After all of the acetonitrile passed through the cartridge, the sampleis spotted onto the center of the freshly washed disc and allowed toadsorb onto the disc for 10 minutes. The disc is washed with 1 mL ofacetonitrile followed by five ishes of 1 mL of 96% acetonitrile. Thecartridges are placed over a 1.5 mL Eppendorf tube and the 2-AB labeledoligosaccharides are eluted with 3 ishes (400 mL each ish) of milli Qwater.

The oligosaccharides are separated using a Glycosep N HPLC (cat#GKI-4728) column connected to a Shimadzu HPLC system. The Shimadzu HPLCsystem consisted of a system controller, degasser, binary pumps,autosampler with a sample cooler, and a fluorescent detector.

Stability at Elevated Temperatures

The buffer of antibody is either 5.57 mM sodium phosphate monobasic,8.69 mM sodium phosphate dibasic, 106.69 mM NaCl, 1.07 mM sodiumcitrate, 6.45 mM citric acid, 66.68 mM mannitol, 0.1% (w/v) Tween, pH5.2; or 10 mM histidine, 10 mM methionine, 4% mannitol, pH 5.9 usingAmicon ultra centrifugal filters. The final concentration of theantibodies is adjusted to 2 mg/mL with the appropriate buffers. Theantibody solutions are then filter sterized and 0.25 mL aliquots areprepared under sterile conditions. The aliquots are left at either −80°C., 5° C., 25° C., or 40° C. for 1, 2 or 3 weeks. At the end of theincubation period, the samples are analyzed by size exclusionchromatography and SDS-PAGE.

The stability samples are analyzed by SDS-PAGE under both reducing andnon-reducing conditions. The procedure used is the same as describedherein. The gels are stained overnight with colloidal blue stain(Invitrogen cat #46-7015, 46-7016) and destained with Milli-Q wateruntil the background is clear. The stained gels are then scanned usingan Epson Expression scanner (model 1680, S/N DASX003641). To obtain moresensitivity, the same gels are silver stained using silver staining kit(Owl Scientific) and the recommended procedures given by themanufacturer is used.

Example 1.2.2.3.C Efficacy of a Humanized Monoclonal Antibody by Itselfor in Combination with Chemotherapy on the Growth of Human CarcinomaXenografts

Human cancer cells are grown in vitro to 99% viability, 85% confluencein tissue culture flasks. SCID female or male mice (Charles Rivers Labs)at 19-25 grams, are ear tagged and shaved. Mice are then inoculatedsubcutaneously into the right flank with 0.2 ml of 2×10⁶ human tumorcells (1:1 matrigel) on study day 0. Administration (IP, Q3D/week) ofvehicle (PBS), humanized antibody, and/or chemotherapy is initiatedafter mice are size matched into separate cages of mice with mean tumorvolumes of approximately 150 to 200 mm³. The tumors are measured by apair of calipers twice a week starting on approximately day 10 postinoculation and the tumor volumes calculated according to the formulaV=L×W²/2 (V: volume, mm³; L: length, mm; W: width, m) Reduction in tumorvolume is seen in animals treated with mAb alone or in combination withchemotherapy relative to tumors in animals that received only vehicle oran isotype control mAb.

Example 1.2.2.3.D FACS Based Redirected Cytotoxicity (rCTL) Assay

Human CD3+ T cells are isolated from previously frozen isolatedperipheral blood mononuclear cells (PBMC) by a negative selectionenrichment column (R&D Systems, Minneapolis, Minn.; Cat. #HTCC-525). Tcells are stimulated for 4 days in flasks (vent cap, Corning, Acton,Mass.) coated with 10 μg/mL anti-CD3 (OKT-3, eBioscience, Inc., SanDiego, Calif.) and 2 μg/mL anti-CD28 (CD28.2, eBioscience, Inc., SanDiego, Calif.) in D-PBS (Invitrogen, Carlsbad, Calif.) and cultured in30 U/mL IL-2 (Roche) in complete RPMI 1640 media (Invitrogen, Carlsbad,Calif.) with L-glutamine, 55 mM β-ME, Pen/Strep, 10% FBS). T cells arethen rested overnight in 30 U/mL IL-2 before using in assay. DoHH2 orRaji target cells are labeled with PKH26 (Sigma-Aldrich, St. Louis, Mo.)according to manufacturer's instructions. RPMI 1640 media (no phenol,Invitrogen, Carlsbad, Calif.) containing L-glutamine and 10% FBS(Hyclone, Logan, Utah) is used throughout the rCTL assay. (See Dreier etal. (2002) Int. J. Cancer 100:690).

Effector T cells (E) and targets (T) are plated at a final cellconcentration of 10⁵ and 10⁴ cells/well in 96-well plates (Costar #3799,Acton, Mass.), respectively to give an E:T ratio of 10:1. DVD-Igmolecules are diluted to obtain concentration-dependent titrationcurves. After an overnight incubation cells are pelleted and washed withD-PBS once before resuspending in FACS buffer containing 0.1% BSA(Invitrogen, Carlsbad, Calif.), 0.1% sodium azide and 0.5 μg/mLpropidium iodide (BD) in D-PBS. FACS data is collected on a FACS CantoII machine (Becton Dickinson, San Jose, Calif.) and analyzed in Flowjo(Treestar). The percent live targets in the DVD-Ig molecule treatedsamples divided by the percent total targets (control, no treatment) iscalculated to determine percent specific lysis. IC₅₀s are calculated inPrism (Graphpad).

Example 1.4 Generation of a DVD-Ig Molecule

DVD-Ig molecules capable of binding two antigens are constructed usingtwo parent monoclonal antibodies, one against human antigen A, and theother against human antigen B, selected as described herein.

Example 1.4.1 Generation of A DVD-Ig Molecule Having Two Linker Lengths

A constant region containing μl Fc with mutations at 234, and 235 toeliminate ADCC/CDC effector functions is used. Four different anti-A/BDVD-Ig constructs are generated: 2 with short linker and 2 with longlinker, each in two different domain orientations: V_(A)-V_(B)-C andV_(B)-V_(A)-C (see Table 13). The linker sequences, derived from theN-terminal sequence of human Cl/Ck or CH1 domain, are as follows:

For DVDAB constructs:

light chain (if anti-A has λ): Short linker: QPKAAP (SEQ ID NO: 15);Long linker:

(SEQ ID NO: 16) QPKAAPSVTLFPP

light chain (if anti-A has κ): Short linker: TVAAP (SEQ ID NO: 13); Longlinker:

(SEQ ID NO: 14) TVAAPSVFIFPP

heavy chain (γl): Short linker: ASTKGP (SEQ ID NO: 21); Long linker:

(SEQ ID NO: 22) ASTKGPSVFPLAP

For DVDBA constructs:

light chain (if anti-B has λ): Short linker: QPKAAP (SEQ ID NO: 15);Long linker:

(SEQ ID NO: 16) QPKAAPSVTLFPP

light chain (if anti-B has k): Short linker: TVAAP (SEQ ID NO: 13); Longlinker:

(SEQ ID NO: 14) TVAAPSVFIFPP

heavy chain (γl): Short linker: ASTKGP (SEQ ID NO: 21); Long linker:

(SEQ ID NO: 22) ASTKGPSVFPLAP

Heavy and light chain constructs are subcloned into the pBOS expressionvector, and expressed in COS cells, followed by purification by ProteinA chromatography. The purified materials are subjected to SDS-PAGE andSEC analysis.

Table 13 describes the heavy chain and light chain constructs used toexpress each anti-A/B DVD-Ig protein.

TABLE 13 Anti-A/B DVD-Ig Constructs DVD-Ig protein Heavy chain constructLight chain construct DVDABSL DVDABHC-SL DVDABLC-SL DVDABLL DVDABHC-LLDVDABLC-LL DVDBASL DVDBAHC-SL DVDBALC-SL DVDBALL DVDBAHC-LL DVDBALC-LL

Example 1.4.2 Molecular cloning of DNA constructs for DVDABSL andDVDABLL

To generate heavy chain constructs DVDABHC-LL and DVDABHC-SL, VH domainof A antibody is PCR amplified using specific primers (3′ primerscontain short/long liner sequence for SL/LL constructs, respectively);meanwhile VH domain of B antibody is amplified using specific primers(5′ primers contains short/long liner sequence for SL/LL constructs,respectively). Both PCR reactions are performed according to standardPCR techniques and procedures. The two PCR products are gel-purified,and used together as overlapping template for the subsequent overlappingPCR reaction. The overlapping PCR products are subcloned into Srf I andSal I double digested pBOS-hCγl,z non-a mammalian expression vector(Abbott) by using standard homologous recombination approach.

To generate light chain constructs DVDABLC-LL and DVDABLC-SL, VL domainof A antibody is PCR amplified using specific primers (3′ primerscontain short/long liner sequence for SL/LL constructs, respectively);meanwhile VL domain of B antibody is amplified using specific primers(5′ primers contains short/long liner sequence for SL/LL constructs,respectively). Both PCR reactions are performed according to standardPCR techniques and procedures. The two PCR products are gel-purified,and used together as overlapping template for the subsequent overlappingPCR reaction using standard PCR conditions. The overlapping PCR productsare subcloned into Srf I and Not I double digested pBOS-hCk mammalianexpression vector (Abbott) by using standard homologous recombinationapproach. Similar approach has been used to generate DVDBASL and DVDBALLas described below:

Example 1.4.3 Molecular Cloning of DNA Constructs for DVDBASL andDVDBALL

To generate heavy chain constructs DVDBAHC-LL and DVDBAHC-SL, VH domainof antibody B is PCR amplified using specific primers (3′ primerscontain short/long liner sequence for SL/LL constructs, respectively);meanwhile VH domain of antibody A is amplified using specific primers(5′ primers contains short/long liner sequence for SL/LL constructs,respectively). Both PCR reactions are performed according to standardPCR techniques and procedures. The two PCR products are gel-purified,and used together as overlapping template for the subsequent overlappingPCR reaction using standard PCR conditions. The overlapping PCR productsare subcloned into Srf I and Sal I double digested pBOS-hCγl,z non-amammalian expression vector (Abbott) by using standard homologousrecombination approach.

To generate light chain constructs DVDBALC-LL and DVDBALC-SL, VL domainof antibody B is PCR amplified using specific primers (3′ primerscontain short/long liner sequence for SL/LL constructs, respectively);meanwhile VL domain of antibody A is amplified using specific primers(5′ primers contains short/long liner sequence for SL/LL constructs,respectively). Both PCR reactions are performed according to standardPCR techniques and procedures. The two PCR products are gel-purified,and used together as overlapping template for the subsequent overlappingPCR reaction using standard PCR conditions. The overlapping PCR productsare subcloned into Srf I and Not I double digested pBOS-hCk mammalianexpression vector (Abbott) by using standard homologous recombinationapproach.

Example 1.4.4 Construction and Expression of Additional DVD-Ig MoleculeExample 1.4.4.1 Preparation of DVD-Ig Vector Constructs

Parent antibody amino acid sequences for specific antibodies, whichrecognize specific antigens or epitopes thereof, for incorporation intoa DVD-Ig molecule can be obtained by preparation of hybridomas asdescribed above or can be obtained by sequencing known antibody proteinsor nucleic acids. In addition, known sequences can be obtained from theliterature. The sequences can be used to synthesize nucleic acids usingstandard DNA synthesis or amplification technologies and assembling thedesired antibody fragments into expression vectors, using standardrecombinant DNA technology, for expression in cells.

For example, nucleic acid codons were determined from amino acidssequences and oligonucleotide DNA was synthesized by Blue HeronBiotechnology, Inc. (www.blueheronbio.com) Bothell, Wash. USA. Theoligonucleotides were assembled into 300-2,000 base pair double-strandedDNA fragments, cloned into a plasmid vector and sequence-verified.Cloned fragments were assembled using an enzymatic process to yield thecomplete gene and subcloned into an expression vector. (See U.S. Pat.Nos. 7,306,914; 7,297,541; 7,279,159; 7,150,969; 20080115243;20080102475; 20080081379; 20080075690; 20080063780; 20080050506;20080038777; 20080022422; 20070289033; 20070287170; 20070254338;20070243194; 20070225227; 20070207171; 20070150976; 20070135620;20070128190; 20070104722; 20070092484; 20070037196; 20070028321;20060172404; 20060162026; 20060153791; 20030215458; 20030157643).

A group of pHybE vectors (U.S. Patent Application Ser. No. 61/021,282)were used for parental antibody and DVD-Ig molecule cloning. V1, derivedfrom pJP183; pHybE-hCgl,z,non-a V2, was used for cloning of antibody andDVD heavy chains with a wildtype constant region. V2, derived frompJP191; pHybE-hCk V2, was used for cloning of antibody and DVD lightchains with a kappa constant region. V3, derived from pJP192; pHybE-hClV2, was used for cloning of antibody and DVDs light chains with a lambdaconstant region. V4, built with a lambda signal peptide and a kappaconstant region, was used for cloning of DVD light chains with alambda-kappa hybrid V domain. V5, built with a kappa signal peptide anda lambda constant region, was used for cloning of DVD light chains witha kappa-lambda hybrid V domain. V7, derived from pJP183;pHybE-hCgl,z,non-a V2, was used for cloning of antibody and DVD heavychains with a (234,235 AA) mutant constant region.

Referring to Table 14, a number of vectors were used in the cloning ofthe parent antibodies and DVD-Ig molecule VH and VL chains.

TABLE 14 Vectors Used to Clone Parent Antibodies and DVD-Ig Molecules IDHeavy chain vector Light chain vector E26.13 V7 V2 E26.35 V7 V2 1B12.1V7 V2 1B12.3 V7 V2 1B12.6 V7 V2 3D12.8 V7 V2 3D12.16 V7 V2 3D12.10/15 V7V2 3D12r16 V7 V2 E26.13-LL- V7 V2 3D12.16 E26.13-LL- V7 V2 3D12.8E26.13-SS- V7 V2 3D12.16 E26.13-SS- V7 V2 3D12.8 E26.13-SS- V7 V23D12.10/15 E26.35-SS- V7 V2 3D12.10/15 E26.35-SS- V7 V2 3D12r161B12.1-SS- V7 V2 3D12r16 1B12.3-SS- V7 V2 3D12r16 1B12.6-SS- V7 V23D12r16

Example 1.4.4.2 Transfection and Expression in 293 Cells

Expression of the reference antibodies and DVD-Ig molecules wasaccomplished by transiently cotransfecting HEK293 (EBNA) cells withplasmids containing the corresponding light-chain (LC) and heavy-chain(HC) nucleic acids. HEK293 (EBNA) cells were propagated in Freestyle 293media (Invitrogen, Carlsbad Calif.) at a 0.5 L-scale in flasks (2 LCorning Cat #431198) shaking in a CO₂ incubator (8% CO₂, 125 RPM, 37°C.). When the cultures reached a density of 1×10⁶ cells/ml, cells weretransfected with transfection complex. Transfection complex was preparedby first mixing 150 μg LC-plasmid and 100 μg HC-plasmid together in 25ml of Freestyle media, followed by the addition of 500 μl PEI stocksolution [stock solution: 1 mg/ml (pH 7.0) Linear 25 kDa PEI,Polysciences Cat #23966]. The transfection complex was mixed byinversion and allowed to incubate at room temperature for 10 minutesprior to being added to the cell culture. Following transfection,cultures continued to be grown in the CO₂ incubator (8% CO₂, 125 RPM,37° C.). Twenty-four hours after transfection, the culture wassupplemented with 25 ml of a 10% Tryptone Ni solution (Organo Technic,La Courneuve France Cat#19553). Nine days after transfection, cells wereremoved from the cultures by centrifugation (16,000 g, 10 minutes), andthe retained supernatant was sterile filtered (Millipore HV DuraporeStericup, 0.45 um) and placed at 4° C. until initiation of thepurification step.

Each antibody or DVD-Ig molecule was individually purified using adisposable 1 ml packed column (packed by Orochem Technologies)containing MabSelect SuRe resin (GE Healthcare). Columns werepre-equilibrated in PBS and then loaded with the harvested 0.55 Lsamples overnight (15 hours) at 1 ml/minute with the flow-through beingrecirculated back into the feed container. Following the loading step,columns were washed with 20 ml PBS and protein was eluted by feedingelution buffer [50 mM Citric acid pH 3.5] at 4 ml/min and collectingfractions (1 ml) in tubes already containing 0.2 ml of 1.5 M Tris pH 8.2(bringing the final pH to approximately 6.0). Fractions containingantibody were pooled based on the chromatograms and dialyzed into thefinal storage buffer [10 mM citric acid, 10 mM Na₂HPO₄, pH 6.0].Following dialysis, samples were filtered through a 0.22 μm Steriflip(Millipore) and the protein concentration was determined by absorbance[Hewlett Packard 8453 diode array spectrophotometer]. SDS-PAGE analysiswas performed on analytical samples (both reduced and non-reduced) toassess final purity, verify the presence of appropriately sized heavy-and light-chain bands, and confirm the absence of significant amounts offree (e.g., uncomplexed) light chain (in the non-reduced samples).

Table 15 contains the yield data for parent antibodies or DVD-Igconstructs expressed as milligrams per liter in 293 cells.

TABLE 15 Transient Expression in Yields of Parent Antibodies and DVD-IgConstructs in 293 Cells N-terminal C-terminal Variable Variable ParentAntibody Domain Domain Expression Yield or DVD-Ig ID (VD) (VD) (mg/L)3D12.8 IL-1alpha (seq. 1) 40 3D12.16 IL-1alpha (seq. 2) 40 3D12.10/15IL-1alpha (seq. 3) 40 3D12r16 IL-1alpha (seq. 4) 9 E26.13 IL-1beta(seq. 1) 72 E26.35 IL-1beta (seq. 2) 40 E26.13-LL- IL-1beta IL-1alpha 03D12.16 (seq. 1) (seq. 2) E26.13-LL- IL-1beta IL-1alpha 0 3D12.8(seq. 1) (seq. 1) E26.13-SS- IL-1beta IL-1alpha 15 3D12.10/15 (seq. 1)(seq. 3) E26.13-SS- IL-1beta IL-1alpha 0 3D12.16 (seq. 1) (seq. 2)E26.13-SS- IL-1beta IL-1alpha 0 3D12.8 (seq. 1) (seq. 1) E26.35-SS-IL-1beta IL-1alpha 10 3D12.10/15 (seq. 2) (seq. 3) E26.35-SS- IL-1betaIL-1alpha 50 3D12r16 (seq. 2) (seq. 4) 1B12.1 IL-1beta 40.5 (seq. 3)1B12.1-SS- IL-1beta IL-1alpha 9.8 3D12r16 (seq. 3) (seq. 4) 1B12.3IL-1beta 56 (seq. 4) 1B12.3-SS- IL-1beta IL-1alpha 22.2 3D12r16 (seq. 4)(seq. 4) 1B12.6 IL-1beta 55.5 (seq. 5) 1B12.6-SS- IL-1beta IL-1alpha 9.83D12r16 (seq. 6) (seq. 4) All DVDs expressed well in 293 cells. DVDscould be easily purified over a protein A column. In most cases >5 mg/Lpurified DVD-Ig molecule could be obtained easily from supernatants of293 cells.

Example 1.4.5 Characterization and Lead Selection of A/B DVD-IgMolecules

The binding affinities of anti-A/B DVD-Ig molecules are analyzed onBiacore against both protein A and protein B. The tetravalent propertyof the DVD-Ig molecule is examined by multiple binding studies onBiacore. Meanwhile, the neutralization potency of the DVD-Ig moleculesfor protein A and protein B are assessed by bioassays, respectively, asdescribed herein. The DVD-Ig molecules that best retain the affinity andpotency of the original parent mAbs are selected for in-depthphysicochemical and bio-analytical (rat PK) characterizations asdescribed herein for each mAb. Based on the collection of analyses, thefinal lead DVD-Ig molecule is advanced into CHO stable cell linedevelopment, and the CHO-derived material is employed in stability,pharmacokinetic and efficacy studies in cynomolgus monkey, andpreformulation activities.

Example 2 Generation and Characterization of Dual Variable DomainImmunoglobulin (DVD-Ig) Molecules

Dual variable domain immunoglobulin (DVD-Ig) molecules using parentantibodies with known amino acid sequences were generated bysynthesizing polynucleotide fragments encoding DVD-Ig variable heavy andDVD-Ig variable light chain sequences and cloning the fragments into apHybC-D2 vector according to Example 1.4.4.1. The DVD-Ig constructs werecloned into and expressed in 293 cells as described in Example 1, 4.4.2.The DVD-Ig protein was purified according to standard methods.Functional characteristics were determined according to the methodsdescribed in Example 1.1.1 and 1.1.2 as indicated. DVD-Ig VH and VLchains for the DVD-Ig molecules of the invention are provided below.

Example 2.1 Generation of IL-1alpha (Seq. 3) and IL-1beta (Seq. 1)DVD-Ig Molecules with Linker Set 1

TABLE 16 DVD Outer Inner SEQ Variable Variable Variable ID Domain DomainDomain Sequence NO Name Name Name 12345678901234567890123456789012345 52E26.13- E26.13 3D1210/ EVQLVESGGGVVQPGRSLRLSCSASGFIFSRYDMS SS- 15WVRQAPGKGLEWVAYISHGGAGTYYPDSVKGRFTI 3D1210/SRDNSKNTLFLQMDSLRPEDTGVYFCARGGVTKGY 15 VHFDVWGQGTPVTVSSASTKGPEVQLVQSGAEVKKPG VSVKVSCKASGYTFTTYGMHWVRQAPGQGLEWMGWINTYTGESTYADDFQGRVTFTLDTSTSTAYMELSS LRSEDTAVYFCARGIYYYGSSYAMNYWGQGTTVTVSS 53 E26.13- E26.13 3D1210/ DIQMTQSPSSLSASVGDRVTITCRASGNIHNYLTW SS- 15YQQTPGKAPKLLIYNAKTLADGVPSRFSGSGSGTD 3D1210/YTFTISSLQPEDIATYYCQHFWSIPYTFGQGTKLQ 15 VLITRTVAAPDIQMTQSPSSLSASVGDRVTITCRASQ DISNMLNWYQQKPGKTPKLLIYYTSRLYPGVPSRFSGSGSGTDYTFTISSLQPEDIATYFCQQGKTLPYA FGQGTKLEIKR

Example 2.2 Generation of IL-1alpha (Seq. 2) and IL-1beta (Seq. 1)DVD-Ig Molecules with Linker Sets 1 and 2

TABLE 17 DVD Outer Inner SEQ Variable Variable Variable ID Domain DomainDomain Sequence NO Name Name Name 12345678901234567890123456789012345 54E26.13- E26.13 3D12.16 EVQLVESGGGVVQPGRSLRLSCSASGFIFSRYDMS SS-WVRQAPGKGLEWVAYISHGGAGTYYPDSVKGRFTI 3D12.16SRDNSKNTLFLQMDSLRPEDTGVYFCARGGVTKGY VHFDVWGQGTPVTVSSASTKGPEIQLVQSGAEVKKPG ASVKVSCKASGYTFTNYGMNWVRQAPGQDLERMAWINTYTGESTYADDFKGRFTFTLDTSTSTAYMELSS LRSEDTAVYFCARGIYYYGSSYAMDYWGQGTTVTVSS 55 E26.13- E26.13 3D12.16 DIQMTQSPSSLSASVGDRVTITCRASGNIHNYLTW SS-YQQTPGKAPKLLIYNAKTLADGVPSRFSGSGSGTD 3D12.16YTFTISSLQPEDIATYYCQHFWSIPYTFGQGTKLQ VLITRTVAAPDIQMTQSPSSLSASVGDRVTITCRASQ DISNCLNWYQQKPGKTPKLLIYYTSRLHSGVPSRFSGSGSGTDYTFTISSLQPEDIATYFCQQGKTLPYA FGQGTKLEIKR 56 E26.13- E26.133D12.16 EVQLVESGGGVVQPGRSLRLSCSASGFIFSRYDMS LL-WVRQAPGKGLEWVAYISHGGAGTYYPDSVKGRFTI 3D12.16SRDNSKNTLFLQMDSLRPEDTGVYFCARGGVTKGY VHFDVWGQGTPVTVSSASTKGPSVFPLAPEIQLVQSG AEVKKPGASVKVSCKASGYTFTNYGMNWVRQAPGQDLERMAWINTYTGESTYADDFKGRFTFTLDTSTST AYMELSSLRSEDTAVYFCARGIYYYGSSYAMDYWGQGTTVTVSS 57 E26.13- E26.13 3D12.16 DIQMTQSPSSLSASVGDRVTITCRASGNIHNYLTWLL- YQQTPGKAPKLLIYNAKTLADGVPSRFSGSGSGTD 3D12.16YTFTISSLQPEDIATYYCQHFWSIPYTFGQGTKLQ VLITRTVAAPSVFIFPPDIQMTQSPSSLSASVGDRVT ITCRASQDISNCLNWYQQKPGKTPKLLIYYTSRLHSGVPSRFSGSGSGTDYTFTISSLQPEDIATYFCQQ GKTLPYAFGQGTKLEIKR

Example 2.3 Generation of IL-1alpha (Seq. 1) and IL-1beta (Seq. 1)DVD-Ig Molecules with Linker Set 1 and 2

TABLE 18 DVD Outer Inner SEQ Variable Variable Variable ID Domain DomainDomain Sequence NO Name Name Name 12345678901234567890123456789012345 58E26.13- E26.13 3D12.8 EVQLVESGGGVVQPGRSLRLSCSASGFIFSRYDMS SS-WVRQAPGKGLEWVAYISHGGAGTYYPDSVKGRFTI 3D12.8SRDNSKNTLFLQMDSLRPEDTGVYFCARGGVTKGY VHFDVWGQGTPVTVSSASTKGPEIQLVQSGSELKKPG ASVKVSCKASGYTFTNYGMNWVRQAPGQDLERMAWINTYTGESTYADDFKGRFVFSLDTSVSTAYLQISS LKAEDTAVYFCARGIYYYGSSYAMDYWGQGTTVTVSS 59 E26.13- E26.13 3D12.8 DIQMTQSPSSLSASVGDRVTITCRASGNIHNYLTW SS-YQQTPGKAPKLLIYNAKTLADGVPSRFSGSGSGTD 3D12.8YTFTISSLQPEDIATYYCQHFWSIPYTFGQGTKLQ VLITRTVAAPDIQMTQSPSSLSASVGDRVTITCRASQ DISNCLNWYQQKPGKTPKLLIYYTSRLHSGVPSRFSGSGSGTDYTFTISSLQPEDIATYFCQQGKTLPYA FGQGTKLEIKR 60 E26.13- E26.13 3D12.8EVQLVESGGGVVQPGRSLRLSCSASGFIFSRYDMS LL-WVRQAPGKGLEWVAYISHGGAGTYYPDSVKGRFTI 3D12.8SRDNSKNTLFLQMDSLRPEDTGVYFCARGGVTKGY VHFDVWGQGTPVTVSSASTKGPSVFPLAPEIQLVQSG SELKKPGASVKVSCKASGYTFTNYGMNWVRQAPGQDLERMAWINTYTGESTYADDFKGRFVFSLDTSVST AYLQISSLKAEDTAVYFCARGIYYYGSSYAMDYWGQGTTVTVSS 61 E26.13- E26.13 3D12.8 DIQMTQSPSSLSASVGDRVTITCRASGNIHNYLTWLL- YQQTPGKAPKLLIYNAKTLADGVPSRFSGSGSGTD 3D12.8YTFTISSLQPEDIATYYCQHFWSIPYTFGQGTKLQ VLITRTVAAPSVFIFPPDIQMTQSPSSLSASVGDRVT ITCRASQDISNCLNWYQQKPGKTPKLLIYYTSRLHSGVPSRFSGSGSGTDYTFTISSLQPEDIATYFCQQ GKTLPYAFGQGTKLEIKR

Example 2.4 Generation of IL-1alpha (Seq. 3) and IL-1beta (Seq. 2)DVD-Ig Molecules with Linker Set 1

TABLE 19 DVD Outer Inner SEQ Variable Variable Variable ID Domain DomainDomain Sequence NO Name Name Name 12345678901234567890123456789012345 62E26.35- E26.35 3D1210/ EVQLVESGGGVVQPGRSLRLSCSASGFIFSRYDMS SS- 15WVRQAPGKGLEWVAYISHGGAGTYYPDSVKGRFTI 3D1210/SRDNSKNTLFLQMDSLRAEDTAVYYCARGGVYKGY 15 VHFDVWGQGTPVTVSSASTKGPEVQLVQSGAEVKKPG VSVKVSCKASGYTFTTYGMHWVRQAPGQGLEWMGWINTYTGESTYADDFQGRVTFTLDTSTSTAYMELSS LRSEDTAVYFCARGIYYYGSSYAMNYWGQGTTVTVSS 63 E26.35- E26.35 3D1210/ DIQMTQSPSSLSASVGDRVTITCRASGNIHNYLTW SS- 15YQQTPGKAPKLLIYNAKTLADGVPSRFSGSGSGTD 3D1210/YTFTISSLQPEDIATYYCQHFWSIPYTFGQGTKLQ 15 VLITRTVAAPDIQMTQSPSSLSASVGDRVTITCRASQ DISNMLNWYQQKPGKTPKLLIYYTSRLYPGVPSRFSGSGSGTDYTFTISSLQPEDIATYFCQQGKTLPYA FGQGTKLEIKR

Example 2.5 Generation of IL-1alpha (Seq. 4) and IL-1beta (Seq. 2)DVD-Ig Molecules with Linker Set 1

TABLE 20 DVD Outer Inner SEQ Variable Variable Variable ID Domain DomainDomain Sequence NO Name Name Name 12345678901234567890123456789012345 64E26.35- E26.35 3D12r16 EVQLVESGGGVVQPGRSLRLSCSASGFIFSRYDMS SS-WVRQAPGKGLEWVAYISHGGAGTYYPDSVKGRFTI 3D12r16SRDNSKNTLFLQMDSLRAEDTAVYYCARGGVYKGY VHFDVWGQGTPVTVSSASTKGPEVQLVQSGAEVKKPG ASVKVSCKASGYTFKYYGMNWVRQAPGQGLERMGWINTYTGQSTYADDFKGRVTFTLDTSTSTAYMELSS LRSEDTAVYYCARDIYYYGSDFAMDYWGQGTTVTVSS 65 E26.35- E26.35 3D12r16 DIQMTQSPSSLSASVGDRVTITCRASGNIHNYLTW SS-YQQTPGKAPKLLIYNAKTLADGVPSRFSGSGSGTD 3D12r16YTFTISSLQPEDIATYYCQHFWSIPYTFGQGTKLQ VLITRTVAAPDIQMTQSPSSLSASVGDRVTITCRASQ DISNMLNWYQQKPGKAPKLLIYYTSRLKPGVPSRFSGSGSGTDYTFTISSLQPEDIATYFCQQGKTAPYT FGQGTKLEIKR

Example 2.6 Generation of IL-1alpha (Seq. 4) and IL-1beta (Seq. 3)DVD-Ig Molecules with Linker Set 1

TABLE 21 DVD Outer Inner SEQ Variable Variable Variable ID Domain DomainDomain Sequence NO Name Name Name 12345678901234567890123456789012345 661B12.1- 1B12.1 3D12r16 EVQLQESGPGLVKPSETLSLTCTVSGFSLSDYGVS SS-WIRQPPGKGLEWLGLIWGGGDTYYNSPLKSRLTIS 3D12r16KDNSKSQVSLKLSSVTAADTAVYYCAKQRTLWGYD VHLYGMDYWGQGTLVTVSSASTKGPEVQLVQSGAEVK KPGASVKVSCKASGYTFKYYGMNWVRQAPGQGLERMGWINTYTGQSTYADDFKGRVTFTLDTSTSTAYME LSSLRSEDTAVYYCARDIYYYGSDFAMDYWGQGTTVTVSS 67 1B12.1- 1B12.1 3D12r16 DTQVTQSPSSLSASVGDRVTITCITSTDIDVDMNW SS-YQQKPGKPPKLLISQGNTLRPGVPSRFSSSGSGTD 3D12r16FTFTISSLQPEDFATYYCLQSDNLPLTFGQGTKLE VLIKRTVAAPDIQMTQSPSSLSASVGDRVTITCRASQ DISNMLNWYQQKPGKAPKLLIYYTSRLKPGVPSRFSGSGSGTDYTFTISSLQPEDIATYFCQQGKTAPYT FGQGTKLEIKR

Example 2.7 Generation of IL-1alpha (Seq. 4) and IL-1beta (Seq. 4)DVD-Ig Molecules with Linker Set 1

TABLE 22 DVD Outer Inner SEQ Variable Variable Variable ID Domain DomainDomain Sequence NO Name Name Name 12345678901234567890123456789012345 681B12.3- 1B12.3 3D12r16 EVQLQESGPGLVKPSETLSLTCTVSGFSLSDYGVS SS-WIRQPPGKGLEWLGLIWGGGDTYYNSPLKSRLTIS 3D12r16KDNSKSQVSLKLSSVTAADTAVYYCAKQRTLWGYD VHLYGMDYWGQGTLVTVSSASTKGPEVQLVQSGAEVK KPGASVKVSCKASGYTFKYYGMNWVRQAPGQGLERMGWINTYTGQSTYADDFKGRVTFTLDTSTSTAYME LSSLRSEDTAVYYCARDIYYYGSDFAMDYWGQGTTVTVSS 69 1B12.3- 1B12.3 3D12r16 DTVVTQSPAFLSVTPGEKVTITCITSTDIDVDMNW SS-YQQKPDQPPKLLISQGNTLRPGVPSRFSSSGSGTD 3D12r16FTFTISSLEAEDAATYYCLQSDNLPLTFGQGTKLE VLIKRTVAAPDIQMTQSPSSLSASVGDRVTITCRASQ DISNMLNWYQQKPGKAPKLLIYYTSRLKPGVPSRFSGSGSGTDYTFTISSLQPEDIATYFCQQGKTAPYT FGQGTKLEIKR

Example 2.8 Generation of IL-1alpha (Seq. 4) and IL-1beta (Seq. 5)DVD-Ig Molecules with Linker Set 1

TABLE 23 DVD Outer Inner SEQ Variable Variable Variable ID Domain DomainDomain Sequence NO Name Name Name 12345678901234567890123456789012345 701B12.6- 1B12.6 3D12r16 EVQLVESGGGLVQPGGSLRLSCAVSGFTLSDYGVS SS-WIRQAPGKGLEWLGLIWGGGDTYYNSPLKSRLTIS 3D12r16KDNSKSTVYLQMNSLRAEDTAVYYCAKQRTLWGYD VHLYGMDYWGQGTLVTVSSASTKGPEVQLVQSGAEVK KPGASVKVSCKASGYTFKYYGMNWVRQAPGQGLERMGWINTYTGQSTYADDFKGRVTFTLDTSTSTAYME LSSLRSEDTAVYYCARDIYYYGSDFAMDYWGQGTTVTVSS 71 1B12.6- 1B12.6 3D12r16 ETTVTQSPSSLSASVGDRVTITCITSTDIDVDMNW SS-YQQKPGKPPKLLISQGNTLRPGVPSRFSSSGSGTD 3D12r16FTFTISSLQPEDFATYYCLQSDNLPLTFGQGTKLE VLIKRTVAAPDIQMTQSPSSLSASVGDRVTITCRASQ DISNMLNWYQQKPGKAPKLLIYYTSRLKPGVPSRFSGSGSGTDYTFTISSLQPEDIATYFCQQGKTAPYT FGQGTKLEIKR

Example 2.9 Cloning Vector Sequences Used to Clone Parent Antibody andDVD-Ig Sequences

TABLE 24 Vector Nucleotide sequences SEQ ID NO name123456789012345678901234567890123456789012345678901 72 V1GCGTCGACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAAAGTTGAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGCGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAATGAGCGGCCGCTCGAGGCCGGCAAGGCCGGATCCCCCGACCTCGACCTCTGGCTAATAAAGGAAATTTATTTTCATTGCAATAGTGTGTTGGAATTTTTTGTGTCTCTCACTCGGAAGGACATATGGGAGGGCAAATCATTTGGTCGAGATCCCTCGGAGATCTCTAGCTAGAGGATCGATCCCCGCCCCGGACGAACTAAACCTGACTACGACATCTCTGCCCCTTCTTCGCGGGGCAGTGCATGTAATCCCTTCAGTTGGTTGGTACAACTTGCCAACTGGGCCCTGTTCCACATGTGACACGGGGGGGGACCAAACACAAAGGGGTTCTCTGACTGTAGTTGACATCCTTATAAATGGATGTGCACATTTGCCAACACTGAGTGGCTTTCATCCTGGAGCAGACTTTGCAGTCTGTGGACTGCAACACAACATTGCCTTTATGTGTAACTCTTGGCTGAAGCTCTTACACCAATGCTGGGGGACATGTACCTCCCAGGGGCCCAGGAAGACTACGGGAGGCTACACCAACGTCAATCAGAGGGGCCTGTGTAGCTACCGATAAGCGGACCCTCAAGAGGGCATTAGCAATAGTGTTTATAAGGCCCCCTTGTTAACCCTAAACGGGTAGCATATGCTTCCCGGGTAGTAGTATATACTATCCAGACTAACCCTAATTCAATAGCATATGTTACCCAACGGGAAGCATATGCTATCGAATTAGGGTTAGTAAAAGGGTCCTAAGGAACAGCGATATCTCCCACCCCATGAGCTGTCACGGTTTTATTTACATGGGGTCAGGATTCCACGAGGGTAGTGAACCATTTTAGTCACAAGGGCAGTGGCTGAAGATCAAGGAGCGGGCAGTGAACTCTCCTGAATCTTCGCCTGCTTCTTCATTCTCCTTCGTTTAGCTAATAGAATAACTGCTGAGTTGTGAACAGTAAGGTGTATGTGAGGTGCTCGAAAACAAGGTTTCAGGTGACGCCCCCAGAATAAAATTTGGACGGGGGGTTCAGTGGTGGCATTGTGCTATGACACCAATATAACCCTCACAAACCCCTTGGGCAATAAATACTAGTGTAGGAATGAAACATTCTGAATATCTTTAACAATAGAAATCCATGGGGTGGGGACAAGCCGTAAAGACTGGATGTCCATCTCACACGAATTTATGGCTATGGGCAACACATAATCCTAGTGCAATATGATACTGGGGTTATTAAGATGTGTCCCAGGCAGGGACCAAGACAGGTGAACCATGTTGTTACACTCTATTTGTAACAAGGGGAAAGAGAGTGGACGCCGACAGCAGCGGACTCCACTGGTTGTCTCTAACACCCCCGAAAATTAAACGGGGCTCCACGCCAATGGGGCCCATAAACAAAGACAAGTGGCCACTCTTTTTTTTGAAATTGTGGAGTGGGGGCACGCGTCAGCCCCCACACGCCGCCCTGCGGTTTTGGACTGTAAAATAAGGGTGTAATAACTTGGCTGATTGTAACCCCGCTAACCACTGCGGTCAAACCACTTGCCCACAAAACCACTAATGGCACCCCGGGGAATACCTGCATAAGTAGGTGGGCGGGCCAAGATAGGGGCGCGATTGCTGCGATCTGGAGGACAAATTACACACACTTGCGCCTGAGCGCCAAGCACAGGGTTGTTGGTCCTCATATTCACGAGGTCGCTGAGAGCACGGTGGGCTAATGTTGCCATGGGTAGCATATACTACCCAAATATCTGGATAGCATATGCTATCCTAATCTATATCTGGGTAGCATAGGCTATCCTAATCTATATCTGGGTAGCATATGCTATCCTAATCTATATCTGGGTAGTATATGCTATCCTAATTTATATCTGGGTAGCATAGGCTATCCTAATCTATATCTGGGTAGCATATGCTATCCTAATCTATATCTGGGTAGTATATGCTATCCTAATCTGTATCCGGGTAGCATATGCTATCCTAATAGAGATTAGGGTAGTATATGCTATCCTAATTTATATCTGGGTAGCATATACTACCCAAATATCTGGATAGCATATGCTATCCTAATCTATATCTGGGTAGCATATGCTATCCTAATCTATATCTGGGTAGCATAGGCTATCCTAATCTATATCTGGGTAGCATATGCTATCCTAATCTATATCTGGGTAGTATATGCTATCCTAATTTATATCTGGGTAGCATAGGCTATCCTAATCTATATCTGGGTAGCATATGCTATCCTAATCTATATCTGGGTAGTATATGCTATCCTAATCTGTATCCGGGTAGCATATGCTATCCTCATGATAAGCTGTCAAACATGAGAATTTTCTTGAAGACGAAAGGGCCTCGTGATACGCCTATTTTTATAGGTTAATGTCATGATAATAATGGTTTCTTAGACGTCAGGTGGCACTTTTCGGGGAAATGTGCGCGGAACCCCTATTTGTTTATTTTTCTAAATACATTCAAATATGTATCCGCTCATGAGACAATAACCCTGATAAATGCTTCAATAATATTGAAAAAGGAAGAGTATGAGTATTCAACATTTCCGTGTCGCCCTTATTCCCTTTTTTGCGGCATTTTGCCTTCCTGTTTTTGCTCACCCAGAAACGCTGGTGAAAGTAAAAGATGCTGAAGATCAGTTGGGTGCACGAGTGGGTTACATCGAACTGGATCTCAACAGCGGTAAGATCCTTGAGAGTTTTCGCCCCGAAGAACGTTTTCCAATGATGAGCACTTTTAAAGTTCTGCTATGTGGCGCGGTATTATCCCGTGTTGACGCCGGGCAAGAGCAACTCGGTCGCCGCATACACTATTCTCAGAATGACTTGGTTGAGTACTCACCAGTCACAGAAAAGCATCTTACGGATGGCATGACAGTAAGAGAATTATGCAGTGCTGCCATAACCATGAGTGATAACACTGCGGCCAACTTACTTCTGACAACGATCGGAGGACCGAAGGAGCTAACCGCTTTTTTGCACAACATGGGGGATCATGTAACTCGCCTTGATCGTTGGGAACCGGAGCTGAATGAAGCCATACCAAACGACGAGCGTGACACCACGATGCCTGCAGCAATGGCAACAACGTTGCGCAAACTATTAACTGGCGAACTACTTACTCTAGCTTCCCGGCAACAATTAATAGACTGGATGGAGGCGGATAAAGTTGCAGGACCACTTCTGCGCTCGGCCCTTCCGGCTGGCTGGTTTATTGCTGATAAATCTGGAGCCGGTGAGCGTGGGTCTCGCGGTATCATTGCAGCACTGGGGCCAGATGGTAAGCCCTCCCGTATCGTAGTTATCTACACGACGGGGAGTCAGGCAACTATGGATGAACGAAATAGACAGATCGCTGAGATAGGTGCCTCACTGATTAAGCATTGGTAACTGTCAGACCAAGTTTACTCATATATACTTTAGATTGATTTAAAACTTCATTTTTAATTTAAAAGGATCTAGGTGAAGATCCTTTTTGATAATCTCATGACCAAAATCCCTTAACGTGAGTTTTCGTTCCACTGAGCGTCAGACCCCGTAGAAAAGATCAAAGGATCTTCTTGAGATCCTTTTTTTCTGCGCGTAATCTGCTGCTTGCAAACAAAAAAACCACCGCTACCAGCGGTGGTTTGTTTGCCGGATCAAGAGCTACCAACTCTTTTTCCGAAGGTAACTGGCTTCAGCAGAGCGCAGATACCAAATACTGTTCTTCTAGTGTAGCCGTAGTTAGGCCACCACTTCAAGAACTCTGTAGCACCGCCTACATACCTCGCTCTGCTAATCCTGTTACCAGTGGCTGCTGCCAGTGGCGATAAGTCGTGTCTTACCGGGTTGGACTCAAGACGATAGTTACCGGATAAGGCGCAGCGGTCGGGCTGAACGGGGGGTTCGTGCACACAGCCCAGCTTGGAGCGAACGACCTACACCGAACTGAGATACCTACAGCGTGAGCTATGAGAAAGCGCCACGCTTCCCGAAGGGAGAAAGGCGGACAGGTATCCGGTAAGCGGCAGGGTCGGAACAGGAGAGCGCACGAGGGAGCTTCCAGGGGGAAACGCCTGGTATCTTTATAGTCCTGTCGGGTTTCGCCACCTCTGACTTGAGCGTCGATTTTTGTGATGCTCGTCAGGGGGGCGGAGCCTATGGAAAAACGCCAGCAACGCGGCCTTTTTACGGTTCCTGGCCTTTTGCTGGCCTTTTGCTCACATGTTCTTTCCTGCGTTATCCCCTGATTCTGTGGATAACCGTATTACCGCCTTTGAGTGAGCTGATACCGCTCGCCGCAGCCGAACGACCGAGCGCAGCGAGTCAGTGAGCGAGGAAGCGGAAGAGCGCCCAATACGCAAACCGCCTCTCCCCGCGCGTTGGCCGATTCATTAATGCAGCTGGCACGACAGGTTTCCCGACTGGAAAGCGGGCAGTGAGCGCAACGCAATTAATGTGAGTTAGCTCACTCATTAGGCACCCCAGGCTTTACACTTTATGCTTCCGGCTCGTATGTTGTGTGGAATTGTGAGCGGATAACAATTTCACACAGGAAACAGCTATGACCATGATTACGCCAAGCTCTAGCTAGAGGTCGAGTCCCTCCCCAGCAGGCAGAAGTATGCAAAGCATGCATCTCAATTAGTCAGCAACCATAGTCCCGCCCCTAACTCCGCCCATCCCGCCCCTAACTCCGCCCAGTTCCGCCCATTCTCCGCCCCATGGCTGACTAATTTTTTTTATTTATGCAGAGGCCGAGGCCGCCTCGGCCTCTGAGCTATTCCAGAAGTAGTGAGGAGGCTTTTTTGGAGGCCTAGGCTTTTGCAAAAAGCTTTGCAAAGATGGATAAAGTTTTAAACAGAGAGGAATCTTTGCAGCTAATGGACCTTCTAGGTCTTGAAAGGAGTGGGAATTGGCTCCGGTGCCCGTCAGTGGGCAGAGCGCACATCGCCCACAGTCCCCGAGAAGTTGGGGGGAGGGGTCGGCAATTGAACCGGTGCCTAGAGAAGGTGGCGCGGGGTAAACTGGGAAAGTGATGTCGTGTACTGGCTCCGCCTTTTTCCCGAGGGTGGGGGAGAACCGTATATAAGTGCAGTAGTCGCCGTGAACGTTCTTTTTCGCAACGGGTTTGCCGCCAGAACACAGGTAAGTGCCGTGTGTGGTTCCCGCGGGCCTGGCCTCTTTACGGGTTATGGCCCTTGCGTGCCTTGAATTACTTCCACCTGGCTGCAGTACGTGATTCTTGATCCCGAGCTTCGGGTTGGAAGTGGGTGGGAGAGTTCGAGGCCTTGCGCTTAAGGAGCCCCTTCGCCTCGTGCTTGAGTTGAGGCCTGGCCTGGGCGCTGGGGCCGCCGCGTGCGAATCTGGTGGCACCTTCGCGCCTGTCTCGCTGCTTTCGATAAGTCTCTAGCCATTTAAAATTTTTGATGACCTGCTGCGACGCTTTTTTTCTGGCAAGATAGTCTTGTAAATGCGGGCCAAGATCTGCACACTGGTATTTCGGTTTTTGGGGCCGCGGGCGGCGACGGGGCCCGTGCGTCCCAGCGCACATGTTCGGCGAGGCGGGGCCTGCGAGCGCGGCCACCGAGAATCGGACGGGGGTAGTCTCAAGCTGGCCGGCCTGCTCTGGTGCCTGGCCTCGCGCCGCCGTGTATCGCCCCGCCCTGGGCGGCAAGGCTGGCCCGGTCGGCACCAGTTGCGTGAGCGGAAAGATGGCCGCTTCCCGGCCCTGCTGCAGGGAGCTCAAAATGGAGGACGCGGCGCTCGGGAGAGCGGGCGGGTGAGTCACCCACACAAAGGAAAAGGGCCTTTCCGTCCTCAGCCGTCGCTTCATGTGACTCCACGGAGTACCGGGCGCCGTCCAGGCACCTCGATTAGTTCTCGAGCTTTTGGAGTACGTCGTCTTTAGGTTGGGGGGAGGGGTTTTATGCGATGGAGTTTCCCCACACTGAGTGGGTGGAGACTGAAGTTAGGCCAGCTTGGCACTTGATGTAATTCTCCTTGGAATTTGCCCTTTTTGAGTTTGGATCTTGGTTCATTCTCAAGCCTCAGACAGTGGTTCAAAGTTTTTTTCTTCCATTTCAGGTGTCGTGAGGAATTCTCTAGAGATCCCTCGACCTCGAGATCCATTGTGCCCGGGCGCCACCATGGAGTTTGGGCTGAGCTGGCTTTTTCTTGTCGCGATTTTAAAAGGTGTCCAGTGC 73 V2ACGGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGTTGAGCGGCCGCTCGAGGCCGGCAAGGCCGGATCCCCCGACCTCGACCTCTGGCTAATAAAGGAAATTTATTTTCATTGCAATAGTGTGTTGGAATTTTTTGTGTCTCTCACTCGGAAGGACATATGGGAGGGCAAATCATTTGGTCGAGATCCCTCGGAGATCTCTAGCTAGAGGATCGATCCCCGCCCCGGACGAACTAAACCTGACTACGACATCTCTGCCCCTTCTTCGCGGGGCAGTGCATGTAATCCCTTCAGTTGGTTGGTACAACTTGCCAACTGGGCCCTGTTCCACATGTGACACGGGGGGGGACCAAACACAAAGGGGTTCTCTGACTGTAGTTGACATCCTTATAAATGGATGTGCACATTTGCCAACACTGAGTGGCTTTCATCCTGGAGCAGACTTTGCAGTCTGTGGACTGCAACACAACATTGCCTTTATGTGTAACTCTTGGCTGAAGCTCTTACACCAATGCTGGGGGACATGTACCTCCCAGGGGCCCAGGAAGACTACGGGAGGCTACACCAACGTCAATCAGAGGGGCCTGTGTAGCTACCGATAAGCGGACCCTCAAGAGGGCATTAGCAATAGTGTTTATAAGGCCCCCTTGTTAACCCTAAACGGGTAGCATATGCTTCCCGGGTAGTAGTATATACTATCCAGACTAACCCTAATTCAATAGCATATGTTACCCAACGGGAAGCATATGCTATCGAATTAGGGTTAGTAAAAGGGTCCTAAGGAACAGCGATATCTCCCACCCCATGAGCTGTCACGGTTTTATTTACATGGGGTCAGGATTCCACGAGGGTAGTGAACCATTTTAGTCACAAGGGCAGTGGCTGAAGATCAAGGAGCGGGCAGTGAACTCTCCTGAATCTTCGCCTGCTTCTTCATTCTCCTTCGTTTAGCTAATAGAATAACTGCTGAGTTGTGAACAGTAAGGTGTATGTGAGGTGCTCGAAAACAAGGTTTCAGGTGACGCCCCCAGAATAAAATTTGGACGGGGGGTTCAGTGGTGGCATTGTGCTATGACACCAATATAACCCTCACAAACCCCTTGGGCAATAAATACTAGTGTAGGAATGAAACATTCTGAATATCTTTAACAATAGAAATCCATGGGGTGGGGACAAGCCGTAAAGACTGGATGTCCATCTCACACGAATTTATGGCTATGGGCAACACATAATCCTAGTGCAATATGATACTGGGGTTATTAAGATGTGTCCCAGGCAGGGACCAAGACAGGTGAACCATGTTGTTACACTCTATTTGTAACAAGGGGAAAGAGAGTGGACGCCGACAGCAGCGGACTCCACTGGTTGTCTCTAACACCCCCGAAAATTAAACGGGGCTCCACGCCAATGGGGCCCATAAACAAAGACAAGTGGCCACTCTTTTTTTTGAAATTGTGGAGTGGGGGCACGCGTCAGCCCCCACACGCCGCCCTGCGGTTTTGGACTGTAAAATAAGGGTGTAATAACTTGGCTGATTGTAACCCCGCTAACCACTGCGGTCAAACCACTTGCCCACAAAACCACTAATGGCACCCCGGGGAATACCTGCATAAGTAGGTGGGCGGGCCAAGATAGGGGCGCGATTGCTGCGATCTGGAGGACAAATTACACACACTTGCGCCTGAGCGCCAAGCACAGGGTTGTTGGTCCTCATATTCACGAGGTCGCTGAGAGCACGGTGGGCTAATGTTGCCATGGGTAGCATATACTACCCAAATATCTGGATAGCATATGCTATCCTAATCTATATCTGGGTAGCATAGGCTATCCTAATCTATATCTGGGTAGCATATGCTATCCTAATCTATATCTGGGTAGTATATGCTATCCTAATTTATATCTGGGTAGCATAGGCTATCCTAATCTATATCTGGGTAGCATATGCTATCCTAATCTATATCTGGGTAGTATATGCTATCCTAATCTGTATCCGGGTAGCATATGCTATCCTAATAGAGATTAGGGTAGTATATGCTATCCTAATTTATATCTGGGTAGCATATACTACCCAAATATCTGGATAGCATATGCTATCCTAATCTATATCTGGGTAGCATATGCTATCCTAATCTATATCTGGGTAGCATAGGCTATCCTAATCTATATCTGGGTAGCATATGCTATCCTAATCTATATCTGGGTAGTATATGCTATCCTAATTTATATCTGGGTAGCATAGGCTATCCTAATCTATATCTGGGTAGCATATGCTATCCTAATCTATATCTGGGTAGTATATGCTATCCTAATCTGTATCCGGGTAGCATATGCTATCCTCATGATAAGCTGTCAAACATGAGAATTTTCTTGAAGACGAAAGGGCCTCGTGATACGCCTATTTTTATAGGTTAATGTCATGATAATAATGGTTTCTTAGACGTCAGGTGGCACTTTTCGGGGAAATGTGCGCGGAACCCCTATTTGTTTATTTTTCTAAATACATTCAAATATGTATCCGCTCATGAGACAATAACCCTGATAAATGCTTCAATAATATTGAAAAAGGAAGAGTATGAGTATTCAACATTTCCGTGTCGCCCTTATTCCCTTTTTTGCGGCATTTTGCCTTCCTGTTTTTGCTCACCCAGAAACGCTGGTGAAAGTAAAAGATGCTGAAGATCAGTTGGGTGCACGAGTGGGTTACATCGAACTGGATCTCAACAGCGGTAAGATCCTTGAGAGTTTTCGCCCCGAAGAACGTTTTCCAATGATGAGCACTTTTAAAGTTCTGCTATGTGGCGCGGTATTATCCCGTGTTGACGCCGGGCAAGAGCAACTCGGTCGCCGCATACACTATTCTCAGAATGACTTGGTTGAGTACTCACCAGTCACAGAAAAGCATCTTACGGATGGCATGACAGTAAGAGAATTATGCAGTGCTGCCATAACCATGAGTGATAACACTGCGGCCAACTTACTTCTGACAACGATCGGAGGACCGAAGGAGCTAACCGCTTTTTTGCACAACATGGGGGATCATGTAACTCGCCTTGATCGTTGGGAACCGGAGCTGAATGAAGCCATACCAAACGACGAGCGTGACACCACGATGCCTGCAGCAATGGCAACAACGTTGCGCAAACTATTAACTGGCGAACTACTTACTCTAGCTTCCCGGCAACAATTAATAGACTGGATGGAGGCGGATAAAGTTGCAGGACCACTTCTGCGCTCGGCCCTTCCGGCTGGCTGGTTTATTGCTGATAAATCTGGAGCCGGTGAGCGTGGGTCTCGCGGTATCATTGCAGCACTGGGGCCAGATGGTAAGCCCTCCCGTATCGTAGTTATCTACACGACGGGGAGTCAGGCAACTATGGATGAACGAAATAGACAGATCGCTGAGATAGGTGCCTCACTGATTAAGCATTGGTAACTGTCAGACCAAGTTTACTCATATATACTTTAGATTGATTTAAAACTTCATTTTTAATTTAAAAGGATCTAGGTGAAGATCCTTTTTGATAATCTCATGACCAAAATCCCTTAACGTGAGTTTTCGTTCCACTGAGCGTCAGACCCCGTAGAAAAGATCAAAGGATCTTCTTGAGATCCTTTTTTTCTGCGCGTAATCTGCTGCTTGCAAACAAAAAAACCACCGCTACCAGCGGTGGTTTGTTTGCCGGATCAAGAGCTACCAACTCTTTTTCCGAAGGTAACTGGCTTCAGCAGAGCGCAGATACCAAATACTGTTCTTCTAGTGTAGCCGTAGTTAGGCCACCACTTCAAGAACTCTGTAGCACCGCCTACATACCTCGCTCTGCTAATCCTGTTACCAGTGGCTGCTGCCAGTGGCGATAAGTCGTGTCTTACCGGGTTGGACTCAAGACGATAGTTACCGGATAAGGCGCAGCGGTCGGGCTGAACGGGGGGTTCGTGCACACAGCCCAGCTTGGAGCGAACGACCTACACCGAACTGAGATACCTACAGCGTGAGCTATGAGAAAGCGCCACGCTTCCCGAAGGGAGAAAGGCGGACAGGTATCCGGTAAGCGGCAGGGTCGGAACAGGAGAGCGCACGAGGGAGCTTCCAGGGGGAAACGCCTGGTATCTTTATAGTCCTGTCGGGTTTCGCCACCTCTGACTTGAGCGTCGATTTTTGTGATGCTCGTCAGGGGGGCGGAGCCTATGGAAAAACGCCAGCAACGCGGCCTTTTTACGGTTCCTGGCCTTTTGCTGGCCTTTTGCTCACATGTTCTTTCCTGCGTTATCCCCTGATTCTGTGGATAACCGTATTACCGCCTTTGAGTGAGCTGATACCGCTCGCCGCAGCCGAACGACCGAGCGCAGCGAGTCAGTGAGCGAGGAAGCGGAAGAGCGCCCAATACGCAAACCGCCTCTCCCCGCGCGTTGGCCGATTCATTAATGCAGCTGGCACGACAGGTTTCCCGACTGGAAAGCGGGCAGTGAGCGCAACGCAATTAATGTGAGTTAGCTCACTCATTAGGCACCCCAGGCTTTACACTTTATGCTTCCGGCTCGTATGTTGTGTGGAATTGTGAGCGGATAACAATTTCACACAGGAAACAGCTATGACCATGATTACGCCAAGCTCTAGCTAGAGGTCGAGTCCCTCCCCAGCAGGCAGAAGTATGCAAAGCATGCATCTCAATTAGTCAGCAACCATAGTCCCGCCCCTAACTCCGCCCATCCCGCCCCTAACTCCGCCCAGTTCCGCCCATTCTCCGCCCCATGGCTGACTAATTTTTTTTATTTATGCAGAGGCCGAGGCCGCCTCGGCCTCTGAGCTATTCCAGAAGTAGTGAGGAGGCTTTTTTGGAGGCCTAGGCTTTTGCAAAAAGCTTTGCAAAGATGGATAAAGTTTTAAACAGAGAGGAATCTTTGCAGCTAATGGACCTTCTAGGTCTTGAAAGGAGTGGGAATTGGCTCCGGTGCCCGTCAGTGGGCAGAGCGCACATCGCCCACAGTCCCCGAGAAGTTGGGGGGAGGGGTCGGCAATTGAACCGGTGCCTAGAGAAGGTGGCGCGGGGTAAACTGGGAAAGTGATGTCGTGTACTGGCTCCGCCTTTTTCCCGAGGGTGGGGGAGAACCGTATATAAGTGCAGTAGTCGCCGTGAACGTTCTTTTTCGCAACGGGTTTGCCGCCAGAACACAGGTAAGTGCCGTGTGTGGTTCCCGCGGGCCTGGCCTCTTTACGGGTTATGGCCCTTGCGTGCCTTGAATTACTTCCACCTGGCTGCAGTACGTGATTCTTGATCCCGAGCTTCGGGTTGGAAGTGGGTGGGAGAGTTCGAGGCCTTGCGCTTAAGGAGCCCCTTCGCCTCGTGCTTGAGTTGAGGCCTGGCCTGGGCGCTGGGGCCGCCGCGTGCGAATCTGGTGGCACCTTCGCGCCTGTCTCGCTGCTTTCGATAAGTCTCTAGCCATTTAAAATTTTTGATGACCTGCTGCGACGCTTTTTTTCTGGCAAGATAGTCTTGTAAATGCGGGCCAAGATCTGCACACTGGTATTTCGGTTTTTGGGGCCGCGGGCGGCGACGGGGCCCGTGCGTCCCAGCGCACATGTTCGGCGAGGCGGGGCCTGCGAGCGCGGCCACCGAGAATCGGACGGGGGTAGTCTCAAGCTGGCCGGCCTGCTCTGGTGCCTGGCCTCGCGCCGCCGTGTATCGCCCCGCCCTGGGCGGCAAGGCTGGCCCGGTCGGCACCAGTTGCGTGAGCGGAAAGATGGCCGCTTCCCGGCCCTGCTGCAGGGAGCTCAAAATGGAGGACGCGGCGCTCGGGAGAGCGGGCGGGTGAGTCACCCACACAAAGGAAAAGGGCCTTTCCGTCCTCAGCCGTCGCTTCATGTGACTCCACGGAGTACCGGGCGCCGTCCAGGCACCTCGATTAGTTCTCGAGCTTTTGGAGTACGTCGTCTTTAGGTTGGGGGGAGGGGTTTTATGCGATGGAGTTTCCCCACACTGAGTGGGTGGAGACTGAAGTTAGGCCAGCTTGGCACTTGATGTAATTCTCCTTGGAATTTGCCCTTTTTGAGTTTGGATCTTGGTTCATTCTCAAGCCTCAGACAGTGGTTCAAAGTTTTTTTCTTCCATTTCAGGTGTCGTGAGGAATTCTCTAGAGATCCCTCGACCTCGAGATCCATTGTGCCCGGGCGCACCATGGACATGCGCGTGCCCGCCCAGCTGCTGGGCCTGCTGCTGCTGTGGTTCCCCGGCTCGCGATGC 74 V3CAACCCAAGGCTGCCCCCTCGGTCACTCTGTTCCCGCCCTCCTCTGAGGAGCTTCAAGCCAACAAGGCCACACTGGTGTGTCTCATAAGTGACTTCTACCCGGGAGCCGTGACAGTGGCCTGGAAGGCAGATAGCAGCCCCGTCAAGGCGGGAGTGGAGACCACCACACCCTCCAAACAAAGCAACAACAAGTACGCGGCCAGCAGCTACCTGAGCCTGACGCCTGAGCAGTGGAAGTCCCACAGAAGCTACAGCTGCCAGGTCACGCATGAAGGGAGCACCGTGGAGAAGACAGTGGCCCCTACAGAATGTTCATGAGCGGCCGCTCGAGGCCGGCAAGGCCGGATCCCCCGACCTCGACCTCTGGCTAATAAAGGAAATTTATTTTCATTGCAATAGTGTGTTGGAATTTTTTGTGTCTCTCACTCGGAAGGACATATGGGAGGGCAAATCATTTGGTCGAGATCCCTCGGAGATCTCTAGCTAGAGGATCGATCCCCGCCCCGGACGAACTAAACCTGACTACGACATCTCTGCCCCTTCTTCGCGGGGCAGTGCATGTAATCCCTTCAGTTGGTTGGTACAACTTGCCAACTGGGCCCTGTTCCACATGTGACACGGGGGGGGACCAAACACAAAGGGGTTCTCTGACTGTAGTTGACATCCTTATAAATGGATGTGCACATTTGCCAACACTGAGTGGCTTTCATCCTGGAGCAGACTTTGCAGTCTGTGGACTGCAACACAACATTGCCTTTATGTGTAACTCTTGGCTGAAGCTCTTACACCAATGCTGGGGGACATGTACCTCCCAGGGGCCCAGGAAGACTACGGGAGGCTACACCAACGTCAATCAGAGGGGCCTGTGTAGCTACCGATAAGCGGACCCTCAAGAGGGCATTAGCAATAGTGTTTATAAGGCCCCCTTGTTAACCCTAAACGGGTAGCATATGCTTCCCGGGTAGTAGTATATACTATCCAGACTAACCCTAATTCAATAGCATATGTTACCCAACGGGAAGCATATGCTATCGAATTAGGGTTAGTAAAAGGGTCCTAAGGAACAGCGATATCTCCCACCCCATGAGCTGTCACGGTTTTATTTACATGGGGTCAGGATTCCACGAGGGTAGTGAACCATTTTAGTCACAAGGGCAGTGGCTGAAGATCAAGGAGCGGGCAGTGAACTCTCCTGAATCTTCGCCTGCTTCTTCATTCTCCTTCGTTTAGCTAATAGAATAACTGCTGAGTTGTGAACAGTAAGGTGTATGTGAGGTGCTCGAAAACAAGGTTTCAGGTGACGCCCCCAGAATAAAATTTGGACGGGGGGTTCAGTGGTGGCATTGTGCTATGACACCAATATAACCCTCACAAACCCCTTGGGCAATAAATACTAGTGTAGGAATGAAACATTCTGAATATCTTTAACAATAGAAATCCATGGGGTGGGGACAAGCCGTAAAGACTGGATGTCCATCTCACACGAATTTATGGCTATGGGCAACACATAATCCTAGTGCAATATGATACTGGGGTTATTAAGATGTGTCCCAGGCAGGGACCAAGACAGGTGAACCATGTTGTTACACTCTATTTGTAACAAGGGGAAAGAGAGTGGACGCCGACAGCAGCGGACTCCACTGGTTGTCTCTAACACCCCCGAAAATTAAACGGGGCTCCACGCCAATGGGGCCCATAAACAAAGACAAGTGGCCACTCTTTTTTTTGAAATTGTGGAGTGGGGGCACGCGTCAGCCCCCACACGCCGCCCTGCGGTTTTGGACTGTAAAATAAGGGTGTAATAACTTGGCTGATTGTAACCCCGCTAACCACTGCGGTCAAACCACTTGCCCACAAAACCACTAATGGCACCCCGGGGAATACCTGCATAAGTAGGTGGGCGGGCCAAGATAGGGGCGCGATTGCTGCGATCTGGAGGACAAATTACACACACTTGCGCCTGAGCGCCAAGCACAGGGTTGTTGGTCCTCATATTCACGAGGTCGCTGAGAGCACGGTGGGCTAATGTTGCCATGGGTAGCATATACTACCCAAATATCTGGATAGCATATGCTATCCTAATCTATATCTGGGTAGCATAGGCTATCCTAATCTATATCTGGGTAGCATATGCTATCCTAATCTATATCTGGGTAGTATATGCTATCCTAATTTATATCTGGGTAGCATAGGCTATCCTAATCTATATCTGGGTAGCATATGCTATCCTAATCTATATCTGGGTAGTATATGCTATCCTAATCTGTATCCGGGTAGCATATGCTATCCTAATAGAGATTAGGGTAGTATATGCTATCCTAATTTATATCTGGGTAGCATATACTACCCAAATATCTGGATAGCATATGCTATCCTAATCTATATCTGGGTAGCATATGCTATCCTAATCTATATCTGGGTAGCATAGGCTATCCTAATCTATATCTGGGTAGCATATGCTATCCTAATCTATATCTGGGTAGTATATGCTATCCTAATTTATATCTGGGTAGCATAGGCTATCCTAATCTATATCTGGGTAGCATATGCTATCCTAATCTATATCTGGGTAGTATATGCTATCCTAATCTGTATCCGGGTAGCATATGCTATCCTCATGATAAGCTGTCAAACATGAGAATTTTCTTGAAGACGAAAGGGCCTCGTGATACGCCTATTTTTATAGGTTAATGTCATGATAATAATGGTTTCTTAGACGTCAGGTGGCACTTTTCGGGGAAATGTGCGCGGAACCCCTATTTGTTTATTTTTCTAAATACATTCAAATATGTATCCGCTCATGAGACAATAACCCTGATAAATGCTTCAATAATATTGAAAAAGGAAGAGTATGAGTATTCAACATTTCCGTGTCGCCCTTATTCCCTTTTTTGCGGCATTTTGCCTTCCTGTTTTTGCTCACCCAGAAACGCTGGTGAAAGTAAAAGATGCTGAAGATCAGTTGGGTGCACGAGTGGGTTACATCGAACTGGATCTCAACAGCGGTAAGATCCTTGAGAGTTTTCGCCCCGAAGAACGTTTTCCAATGATGAGCACTTTTAAAGTTCTGCTATGTGGCGCGGTATTATCCCGTGTTGACGCCGGGCAAGAGCAACTCGGTCGCCGCATACACTATTCTCAGAATGACTTGGTTGAGTACTCACCAGTCACAGAAAAGCATCTTACGGATGGCATGACAGTAAGAGAATTATGCAGTGCTGCCATAACCATGAGTGATAACACTGCGGCCAACTTACTTCTGACAACGATCGGAGGACCGAAGGAGCTAACCGCTTTTTTGCACAACATGGGGGATCATGTAACTCGCCTTGATCGTTGGGAACCGGAGCTGAATGAAGCCATACCAAACGACGAGCGTGACACCACGATGCCTGCAGCAATGGCAACAACGTTGCGCAAACTATTAACTGGCGAACTACTTACTCTAGCTTCCCGGCAACAATTAATAGACTGGATGGAGGCGGATAAAGTTGCAGGACCACTTCTGCGCTCGGCCCTTCCGGCTGGCTGGTTTATTGCTGATAAATCTGGAGCCGGTGAGCGTGGGTCTCGCGGTATCATTGCAGCACTGGGGCCAGATGGTAAGCCCTCCCGTATCGTAGTTATCTACACGACGGGGAGTCAGGCAACTATGGATGAACGAAATAGACAGATCGCTGAGATAGGTGCCTCACTGATTAAGCATTGGTAACTGTCAGACCAAGTTTACTCATATATACTTTAGATTGATTTAAAACTTCATTTTTAATTTAAAAGGATCTAGGTGAAGATCCTTTTTGATAATCTCATGACCAAAATCCCTTAACGTGAGTTTTCGTTCCACTGAGCGTCAGACCCCGTAGAAAAGATCAAAGGATCTTCTTGAGATCCTTTTTTTCTGCGCGTAATCTGCTGCTTGCAAACAAAAAAACCACCGCTACCAGCGGTGGTTTGTTTGCCGGATCAAGAGCTACCAACTCTTTTTCCGAAGGTAACTGGCTTCAGCAGAGCGCAGATACCAAATACTGTTCTTCTAGTGTAGCCGTAGTTAGGCCACCACTTCAAGAACTCTGTAGCACCGCCTACATACCTCGCTCTGCTAATCCTGTTACCAGTGGCTGCTGCCAGTGGCGATAAGTCGTGTCTTACCGGGTTGGACTCAAGACGATAGTTACCGGATAAGGCGCAGCGGTCGGGCTGAACGGGGGGTTCGTGCACACAGCCCAGCTTGGAGCGAACGACCTACACCGAACTGAGATACCTACAGCGTGAGCTATGAGAAAGCGCCACGCTTCCCGAAGGGAGAAAGGCGGACAGGTATCCGGTAAGCGGCAGGGTCGGAACAGGAGAGCGCACGAGGGAGCTTCCAGGGGGAAACGCCTGGTATCTTTATAGTCCTGTCGGGTTTCGCCACCTCTGACTTGAGCGTCGATTTTTGTGATGCTCGTCAGGGGGGCGGAGCCTATGGAAAAACGCCAGCAACGCGGCCTTTTTACGGTTCCTGGCCTTTTGCTGGCCTTTTGCTCACATGTTCTTTCCTGCGTTATCCCCTGATTCTGTGGATAACCGTATTACCGCCTTTGAGTGAGCTGATACCGCTCGCCGCAGCCGAACGACCGAGCGCAGCGAGTCAGTGAGCGAGGAAGCGGAAGAGCGCCCAATACGCAAACCGCCTCTCCCCGCGCGTTGGCCGATTCATTAATGCAGCTGGCACGACAGGTTTCCCGACTGGAAAGCGGGCAGTGAGCGCAACGCAATTAATGTGAGTTAGCTCACTCATTAGGCACCCCAGGCTTTACACTTTATGCTTCCGGCTCGTATGTTGTGTGGAATTGTGAGCGGATAACAATTTCACACAGGAAACAGCTATGACCATGATTACGCCAAGCTCTAGCTAGAGGTCGAGTCCCTCCCCAGCAGGCAGAAGTATGCAAAGCATGCATCTCAATTAGTCAGCAACCATAGTCCCGCCCCTAACTCCGCCCATCCCGCCCCTAACTCCGCCCAGTTCCGCCCATTCTCCGCCCCATGGCTGACTAATTTTTTTTATTTATGCAGAGGCCGAGGCCGCCTCGGCCTCTGAGCTATTCCAGAAGTAGTGAGGAGGCTTTTTTGGAGGCCTAGGCTTTTGCAAAAAGCTTTGCAAAGATGGATAAAGTTTTAAACAGAGAGGAATCTTTGCAGCTAATGGACCTTCTAGGTCTTGAAAGGAGTGGGAATTGGCTCCGGTGCCCGTCAGTGGGCAGAGCGCACATCGCCCACAGTCCCCGAGAAGTTGGGGGGAGGGGTCGGCAATTGAACCGGTGCCTAGAGAAGGTGGCGCGGGGTAAACTGGGAAAGTGATGTCGTGTACTGGCTCCGCCTTTTTCCCGAGGGTGGGGGAGAACCGTATATAAGTGCAGTAGTCGCCGTGAACGTTCTTTTTCGCAACGGGTTTGCCGCCAGAACACAGGTAAGTGCCGTGTGTGGTTCCCGCGGGCCTGGCCTCTTTACGGGTTATGGCCCTTGCGTGCCTTGAATTACTTCCACCTGGCTGCAGTACGTGATTCTTGATCCCGAGCTTCGGGTTGGAAGTGGGTGGGAGAGTTCGAGGCCTTGCGCTTAAGGAGCCCCTTCGCCTCGTGCTTGAGTTGAGGCCTGGCCTGGGCGCTGGGGCCGCCGCGTGCGAATCTGGTGGCACCTTCGCGCCTGTCTCGCTGCTTTCGATAAGTCTCTAGCCATTTAAAATTTTTGATGACCTGCTGCGACGCTTTTTTTCTGGCAAGATAGTCTTGTAAATGCGGGCCAAGATCTGCACACTGGTATTTCGGTTTTTGGGGCCGCGGGCGGCGACGGGGCCCGTGCGTCCCAGCGCACATGTTCGGCGAGGCGGGGCCTGCGAGCGCGGCCACCGAGAATCGGACGGGGGTAGTCTCAAGCTGGCCGGCCTGCTCTGGTGCCTGGCCTCGCGCCGCCGTGTATCGCCCCGCCCTGGGCGGCAAGGCTGGCCCGGTCGGCACCAGTTGCGTGAGCGGAAAGATGGCCGCTTCCCGGCCCTGCTGCAGGGAGCTCAAAATGGAGGACGCGGCGCTCGGGAGAGCGGGCGGGTGAGTCACCCACACAAAGGAAAAGGGCCTTTCCGTCCTCAGCCGTCGCTTCATGTGACTCCACGGAGTACCGGGCGCCGTCCAGGCACCTCGATTAGTTCTCGAGCTTTTGGAGTACGTCGTCTTTAGGTTGGGGGGAGGGGTTTTATGCGATGGAGTTTCCCCACACTGAGTGGGTGGAGACTGAAGTTAGGCCAGCTTGGCACTTGATGTAATTCTCCTTGGAATTTGCCCTTTTTGAGTTTGGATCTTGGTTCATTCTCAAGCCTCAGACAGTGGTTCAAAGTTTTTTTCTTCCATTTCAGGTGTCGTGAGGAATTCTCTAGAGATCCCTCGACCTCGAGATCCATTGTGCCCGGGCGCCACCATGACTTGGACCCCACTCCTCTTCCTCACCCTCCTCCTCCACTGCACAGGAAGCTTATCG 75 V4ACGGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGTTGAGCGGCCGCTCGAGGCCGGCAAGGCCGGATCCCCCGACCTCGACCTCTGGCTAATAAAGGAAATTTATTTTCATTGCAATAGTGTGTTGGAATTTTTTGTGTCTCTCACTCGGAAGGACATATGGGAGGGCAAATCATTTGGTCGAGATCCCTCGGAGATCTCTAGCTAGAGGATCGATCCCCGCCCCGGACGAACTAAACCTGACTACGACATCTCTGCCCCTTCTTCGCGGGGCAGTGCATGTAATCCCTTCAGTTGGTTGGTACAACTTGCCAACTGGGCCCTGTTCCACATGTGACACGGGGGGGGACCAAACACAAAGGGGTTCTCTGACTGTAGTTGACATCCTTATAAATGGATGTGCACATTTGCCAACACTGAGTGGCTTTCATCCTGGAGCAGACTTTGCAGTCTGTGGACTGCAACACAACATTGCCTTTATGTGTAACTCTTGGCTGAAGCTCTTACACCAATGCTGGGGGACATGTACCTCCCAGGGGCCCAGGAAGACTACGGGAGGCTACACCAACGTCAATCAGAGGGGCCTGTGTAGCTACCGATAAGCGGACCCTCAAGAGGGCATTAGCAATAGTGTTTATAAGGCCCCCTTGTTAACCCTAAACGGGTAGCATATGCTTCCCGGGTAGTAGTATATACTATCCAGACTAACCCTAATTCAATAGCATATGTTACCCAACGGGAAGCATATGCTATCGAATTAGGGTTAGTAAAAGGGTCCTAAGGAACAGCGATATCTCCCACCCCATGAGCTGTCACGGTTTTATTTACATGGGGTCAGGATTCCACGAGGGTAGTGAACCATTTTAGTCACAAGGGCAGTGGCTGAAGATCAAGGAGCGGGCAGTGAACTCTCCTGAATCTTCGCCTGCTTCTTCATTCTCCTTCGTTTAGCTAATAGAATAACTGCTGAGTTGTGAACAGTAAGGTGTATGTGAGGTGCTCGAAAACAAGGTTTCAGGTGACGCCCCCAGAATAAAATTTGGACGGGGGGTTCAGTGGTGGCATTGTGCTATGACACCAATATAACCCTCACAAACCCCTTGGGCAATAAATACTAGTGTAGGAATGAAACATTCTGAATATCTTTAACAATAGAAATCCATGGGGTGGGGACAAGCCGTAAAGACTGGATGTCCATCTCACACGAATTTATGGCTATGGGCAACACATAATCCTAGTGCAATATGATACTGGGGTTATTAAGATGTGTCCCAGGCAGGGACCAAGACAGGTGAACCATGTTGTTACACTCTATTTGTAACAAGGGGAAAGAGAGTGGACGCCGACAGCAGCGGACTCCACTGGTTGTCTCTAACACCCCCGAAAATTAAACGGGGCTCCACGCCAATGGGGCCCATAAACAAAGACAAGTGGCCACTCTTTTTTTTGAAATTGTGGAGTGGGGGCACGCGTCAGCCCCCACACGCCGCCCTGCGGTTTTGGACTGTAAAATAAGGGTGTAATAACTTGGCTGATTGTAACCCCGCTAACCACTGCGGTCAAACCACTTGCCCACAAAACCACTAATGGCACCCCGGGGAATACCTGCATAAGTAGGTGGGCGGGCCAAGATAGGGGCGCGATTGCTGCGATCTGGAGGACAAATTACACACACTTGCGCCTGAGCGCCAAGCACAGGGTTGTTGGTCCTCATATTCACGAGGTCGCTGAGAGCACGGTGGGCTAATGTTGCCATGGGTAGCATATACTACCCAAATATCTGGATAGCATATGCTATCCTAATCTATATCTGGGTAGCATAGGCTATCCTAATCTATATCTGGGTAGCATATGCTATCCTAATCTATATCTGGGTAGTATATGCTATCCTAATTTATATCTGGGTAGCATAGGCTATCCTAATCTATATCTGGGTAGCATATGCTATCCTAATCTATATCTGGGTAGTATATGCTATCCTAATCTGTATCCGGGTAGCATATGCTATCCTAATAGAGATTAGGGTAGTATATGCTATCCTAATTTATATCTGGGTAGCATATACTACCCAAATATCTGGATAGCATATGCTATCCTAATCTATATCTGGGTAGCATATGCTATCCTAATCTATATCTGGGTAGCATAGGCTATCCTAATCTATATCTGGGTAGCATATGCTATCCTAATCTATATCTGGGTAGTATATGCTATCCTAATTTATATCTGGGTAGCATAGGCTATCCTAATCTATATCTGGGTAGCATATGCTATCCTAATCTATATCTGGGTAGTATATGCTATCCTAATCTGTATCCGGGTAGCATATGCTATCCTCATGATAAGCTGTCAAACATGAGAATTTTCTTGAAGACGAAAGGGCCTCGTGATACGCCTATTTTTATAGGTTAATGTCATGATAATAATGGTTTCTTAGACGTCAGGTGGCACTTTTCGGGGAAATGTGCGCGGAACCCCTATTTGTTTATTTTTCTAAATACATTCAAATATGTATCCGCTCATGAGACAATAACCCTGATAAATGCTTCAATAATATTGAAAAAGGAAGAGTATGAGTATTCAACATTTCCGTGTCGCCCTTATTCCCTTTTTTGCGGCATTTTGCCTTCCTGTTTTTGCTCACCCAGAAACGCTGGTGAAAGTAAAAGATGCTGAAGATCAGTTGGGTGCACGAGTGGGTTACATCGAACTGGATCTCAACAGCGGTAAGATCCTTGAGAGTTTTCGCCCCGAAGAACGTTTTCCAATGATGAGCACTTTTAAAGTTCTGCTATGTGGCGCGGTATTATCCCGTGTTGACGCCGGGCAAGAGCAACTCGGTCGCCGCATACACTATTCTCAGAATGACTTGGTTGAGTACTCACCAGTCACAGAAAAGCATCTTACGGATGGCATGACAGTAAGAGAATTATGCAGTGCTGCCATAACCATGAGTGATAACACTGCGGCCAACTTACTTCTGACAACGATCGGAGGACCGAAGGAGCTAACCGCTTTTTTGCACAACATGGGGGATCATGTAACTCGCCTTGATCGTTGGGAACCGGAGCTGAATGAAGCCATACCAAACGACGAGCGTGACACCACGATGCCTGCAGCAATGGCAACAACGTTGCGCAAACTATTAACTGGCGAACTACTTACTCTAGCTTCCCGGCAACAATTAATAGACTGGATGGAGGCGGATAAAGTTGCAGGACCACTTCTGCGCTCGGCCCTTCCGGCTGGCTGGTTTATTGCTGATAAATCTGGAGCCGGTGAGCGTGGGTCTCGCGGTATCATTGCAGCACTGGGGCCAGATGGTAAGCCCTCCCGTATCGTAGTTATCTACACGACGGGGAGTCAGGCAACTATGGATGAACGAAATAGACAGATCGCTGAGATAGGTGCCTCACTGATTAAGCATTGGTAACTGTCAGACCAAGTTTACTCATATATACTTTAGATTGATTTAAAACTTCATTTTTAATTTAAAAGGATCTAGGTGAAGATCCTTTTTGATAATCTCATGACCAAAATCCCTTAACGTGAGTTTTCGTTCCACTGAGCGTCAGACCCCGTAGAAAAGATCAAAGGATCTTCTTGAGATCCTTTTTTTCTGCGCGTAATCTGCTGCTTGCAAACAAAAAAACCACCGCTACCAGCGGTGGTTTGTTTGCCGGATCAAGAGCTACCAACTCTTTTTCCGAAGGTAACTGGCTTCAGCAGAGCGCAGATACCAAATACTGTTCTTCTAGTGTAGCCGTAGTTAGGCCACCACTTCAAGAACTCTGTAGCACCGCCTACATACCTCGCTCTGCTAATCCTGTTACCAGTGGCTGCTGCCAGTGGCGATAAGTCGTGTCTTACCGGGTTGGACTCAAGACGATAGTTACCGGATAAGGCGCAGCGGTCGGGCTGAACGGGGGGTTCGTGCACACAGCCCAGCTTGGAGCGAACGACCTACACCGAACTGAGATACCTACAGCGTGAGCTATGAGAAAGCGCCACGCTTCCCGAAGGGAGAAAGGCGGACAGGTATCCGGTAAGCGGCAGGGTCGGAACAGGAGAGCGCACGAGGGAGCTTCCAGGGGGAAACGCCTGGTATCTTTATAGTCCTGTCGGGTTTCGCCACCTCTGACTTGAGCGTCGATTTTTGTGATGCTCGTCAGGGGGGCGGAGCCTATGGAAAAACGCCAGCAACGCGGCCTTTTTACGGTTCCTGGCCTTTTGCTGGCCTTTTGCTCACATGTTCTTTCCTGCGTTATCCCCTGATTCTGTGGATAACCGTATTACCGCCTTTGAGTGAGCTGATACCGCTCGCCGCAGCCGAACGACCGAGCGCAGCGAGTCAGTGAGCGAGGAAGCGGAAGAGCGCCCAATACGCAAACCGCCTCTCCCCGCGCGTTGGCCGATTCATTAATGCAGCTGGCACGACAGGTTTCCCGACTGGAAAGCGGGCAGTGAGCGCAACGCAATTAATGTGAGTTAGCTCACTCATTAGGCACCCCAGGCTTTACACTTTATGCTTCCGGCTCGTATGTTGTGTGGAATTGTGAGCGGATAACAATTTCACACAGGAAACAGCTATGACCATGATTACGCCAAGCTCTAGCTAGAGGTCGAGTCCCTCCCCAGCAGGCAGAAGTATGCAAAGCATGCATCTCAATTAGTCAGCAACCATAGTCCCGCCCCTAACTCCGCCCATCCCGCCCCTAACTCCGCCCAGTTCCGCCCATTCTCCGCCCCATGGCTGACTAATTTTTTTTATTTATGCAGAGGCCGAGGCCGCCTCGGCCTCTGAGCTATTCCAGAAGTAGTGAGGAGGCTTTTTTGGAGGCCTAGGCTTTTGCAAAAAGCTTTGCAAAGATGGATAAAGTTTTAAACAGAGAGGAATCTTTGCAGCTAATGGACCTTCTAGGTCTTGAAAGGAGTGGGAATTGGCTCCGGTGCCCGTCAGTGGGCAGAGCGCACATCGCCCACAGTCCCCGAGAAGTTGGGGGGAGGGGTCGGCAATTGAACCGGTGCCTAGAGAAGGTGGCGCGGGGTAAACTGGGAAAGTGATGTCGTGTACTGGCTCCGCCTTTTTCCCGAGGGTGGGGGAGAACCGTATATAAGTGCAGTAGTCGCCGTGAACGTTCTTTTTCGCAACGGGTTTGCCGCCAGAACACAGGTAAGTGCCGTGTGTGGTTCCCGCGGGCCTGGCCTCTTTACGGGTTATGGCCCTTGCGTGCCTTGAATTACTTCCACCTGGCTGCAGTACGTGATTCTTGATCCCGAGCTTCGGGTTGGAAGTGGGTGGGAGAGTTCGAGGCCTTGCGCTTAAGGAGCCCCTTCGCCTCGTGCTTGAGTTGAGGCCTGGCCTGGGCGCTGGGGCCGCCGCGTGCGAATCTGGTGGCACCTTCGCGCCTGTCTCGCTGCTTTCGATAAGTCTCTAGCCATTTAAAATTTTTGATGACCTGCTGCGACGCTTTTTTTCTGGCAAGATAGTCTTGTAAATGCGGGCCAAGATCTGCACACTGGTATTTCGGTTTTTGGGGCCGCGGGCGGCGACGGGGCCCGTGCGTCCCAGCGCACATGTTCGGCGAGGCGGGGCCTGCGAGCGCGGCCACCGAGAATCGGACGGGGGTAGTCTCAAGCTGGCCGGCCTGCTCTGGTGCCTGGCCTCGCGCCGCCGTGTATCGCCCCGCCCTGGGCGGCAAGGCTGGCCCGGTCGGCACCAGTTGCGTGAGCGGAAAGATGGCCGCTTCCCGGCCCTGCTGCAGGGAGCTCAAAATGGAGGACGCGGCGCTCGGGAGAGCGGGCGGGTGAGTCACCCACACAAAGGAAAAGGGCCTTTCCGTCCTCAGCCGTCGCTTCATGTGACTCCACGGAGTACCGGGCGCCGTCCAGGCACCTCGATTAGTTCTCGAGCTTTTGGAGTACGTCGTCTTTAGGTTGGGGGGAGGGGTTTTATGCGATGGAGTTTCCCCACACTGAGTGGGTGGAGACTGAAGTTAGGCCAGCTTGGCACTTGATGTAATTCTCCTTGGAATTTGCCCTTTTTGAGTTTGGATCTTGGTTCATTCTCAAGCCTCAGACAGTGGTTCAAAGTTTTTTTCTTCCATTTCAGGTGTCGTGAGGAATTCTCTAGAGATCCCTCGACCTCGAGATCCATTGTGCCCGGGCGCACCATGACTTGGACCCCACTCCTCTTCCTCACCCTCCTCCTCCACTGCACAGGAAGCTTATCG 76 V5CAACCCAAGGCTGCCCCCTCGGTCACTCTGTTCCCGCCCTCCTCTGAGGAGCTTCAAGCCAACAAGGCCACACTGGTGTGTCTCATAAGTGACTTCTACCCGGGAGCCGTGACAGTGGCCTGGAAGGCAGATAGCAGCCCCGTCAAGGCGGGAGTGGAGACCACCACACCCTCCAAACAAAGCAACAACAAGTACGCGGCCAGCAGCTACCTGAGCCTGACGCCTGAGCAGTGGAAGTCCCACAGAAGCTACAGCTGCCAGGTCACGCATGAAGGGAGCACCGTGGAGAAGACAGTGGCCCCTACAGAATGTTCATGAGCGGCCGCTCGAGGCCGGCAAGGCCGGATCCCCCGACCTCGACCTCTGGCTAATAAAGGAAATTTATTTTCATTGCAATAGTGTGTTGGAATTTTTTGTGTCTCTCACTCGGAAGGACATATGGGAGGGCAAATCATTTGGTCGAGATCCCTCGGAGATCTCTAGCTAGAGGATCGATCCCCGCCCCGGACGAACTAAACCTGACTACGACATCTCTGCCCCTTCTTCGCGGGGCAGTGCATGTAATCCCTTCAGTTGGTTGGTACAACTTGCCAACTGGGCCCTGTTCCACATGTGACACGGGGGGGGACCAAACACAAAGGGGTTCTCTGACTGTAGTTGACATCCTTATAAATGGATGTGCACATTTGCCAACACTGAGTGGCTTTCATCCTGGAGCAGACTTTGCAGTCTGTGGACTGCAACACAACATTGCCTTTATGTGTAACTCTTGGCTGAAGCTCTTACACCAATGCTGGGGGACATGTACCTCCCAGGGGCCCAGGAAGACTACGGGAGGCTACACCAACGTCAATCAGAGGGGCCTGTGTAGCTACCGATAAGCGGACCCTCAAGAGGGCATTAGCAATAGTGTTTATAAGGCCCCCTTGTTAACCCTAAACGGGTAGCATATGCTTCCCGGGTAGTAGTATATACTATCCAGACTAACCCTAATTCAATAGCATATGTTACCCAACGGGAAGCATATGCTATCGAATTAGGGTTAGTAAAAGGGTCCTAAGGAACAGCGATATCTCCCACCCCATGAGCTGTCACGGTTTTATTTACATGGGGTCAGGATTCCACGAGGGTAGTGAACCATTTTAGTCACAAGGGCAGTGGCTGAAGATCAAGGAGCGGGCAGTGAACTCTCCTGAATCTTCGCCTGCTTCTTCATTCTCCTTCGTTTAGCTAATAGAATAACTGCTGAGTTGTGAACAGTAAGGTGTATGTGAGGTGCTCGAAAACAAGGTTTCAGGTGACGCCCCCAGAATAAAATTTGGACGGGGGGTTCAGTGGTGGCATTGTGCTATGACACCAATATAACCCTCACAAACCCCTTGGGCAATAAATACTAGTGTAGGAATGAAACATTCTGAATATCTTTAACAATAGAAATCCATGGGGTGGGGACAAGCCGTAAAGACTGGATGTCCATCTCACACGAATTTATGGCTATGGGCAACACATAATCCTAGTGCAATATGATACTGGGGTTATTAAGATGTGTCCCAGGCAGGGACCAAGACAGGTGAACCATGTTGTTACACTCTATTTGTAACAAGGGGAAAGAGAGTGGACGCCGACAGCAGCGGACTCCACTGGTTGTCTCTAACACCCCCGAAAATTAAACGGGGCTCCACGCCAATGGGGCCCATAAACAAAGACAAGTGGCCACTCTTTTTTTTGAAATTGTGGAGTGGGGGCACGCGTCAGCCCCCACACGCCGCCCTGCGGTTTTGGACTGTAAAATAAGGGTGTAATAACTTGGCTGATTGTAACCCCGCTAACCACTGCGGTCAAACCACTTGCCCACAAAACCACTAATGGCACCCCGGGGAATACCTGCATAAGTAGGTGGGCGGGCCAAGATAGGGGCGCGATTGCTGCGATCTGGAGGACAAATTACACACACTTGCGCCTGAGCGCCAAGCACAGGGTTGTTGGTCCTCATATTCACGAGGTCGCTGAGAGCACGGTGGGCTAATGTTGCCATGGGTAGCATATACTACCCAAATATCTGGATAGCATATGCTATCCTAATCTATATCTGGGTAGCATAGGCTATCCTAATCTATATCTGGGTAGCATATGCTATCCTAATCTATATCTGGGTAGTATATGCTATCCTAATTTATATCTGGGTAGCATAGGCTATCCTAATCTATATCTGGGTAGCATATGCTATCCTAATCTATATCTGGGTAGTATATGCTATCCTAATCTGTATCCGGGTAGCATATGCTATCCTAATAGAGATTAGGGTAGTATATGCTATCCTAATTTATATCTGGGTAGCATATACTACCCAAATATCTGGATAGCATATGCTATCCTAATCTATATCTGGGTAGCATATGCTATCCTAATCTATATCTGGGTAGCATAGGCTATCCTAATCTATATCTGGGTAGCATATGCTATCCTAATCTATATCTGGGTAGTATATGCTATCCTAATTTATATCTGGGTAGCATAGGCTATCCTAATCTATATCTGGGTAGCATATGCTATCCTAATCTATATCTGGGTAGTATATGCTATCCTAATCTGTATCCGGGTAGCATATGCTATCCTCATGATAAGCTGTCAAACATGAGAATTTTCTTGAAGACGAAAGGGCCTCGTGATACGCCTATTTTTATAGGTTAATGTCATGATAATAATGGTTTCTTAGACGTCAGGTGGCACTTTTCGGGGAAATGTGCGCGGAACCCCTATTTGTTTATTTTTCTAAATACATTCAAATATGTATCCGCTCATGAGACAATAACCCTGATAAATGCTTCAATAATATTGAAAAAGGAAGAGTATGAGTATTCAACATTTCCGTGTCGCCCTTATTCCCTTTTTTGCGGCATTTTGCCTTCCTGTTTTTGCTCACCCAGAAACGCTGGTGAAAGTAAAAGATGCTGAAGATCAGTTGGGTGCACGAGTGGGTTACATCGAACTGGATCTCAACAGCGGTAAGATCCTTGAGAGTTTTCGCCCCGAAGAACGTTTTCCAATGATGAGCACTTTTAAAGTTCTGCTATGTGGCGCGGTATTATCCCGTGTTGACGCCGGGCAAGAGCAACTCGGTCGCCGCATACACTATTCTCAGAATGACTTGGTTGAGTACTCACCAGTCACAGAAAAGCATCTTACGGATGGCATGACAGTAAGAGAATTATGCAGTGCTGCCATAACCATGAGTGATAACACTGCGGCCAACTTACTTCTGACAACGATCGGAGGACCGAAGGAGCTAACCGCTTTTTTGCACAACATGGGGGATCATGTAACTCGCCTTGATCGTTGGGAACCGGAGCTGAATGAAGCCATACCAAACGACGAGCGTGACACCACGATGCCTGCAGCAATGGCAACAACGTTGCGCAAACTATTAACTGGCGAACTACTTACTCTAGCTTCCCGGCAACAATTAATAGACTGGATGGAGGCGGATAAAGTTGCAGGACCACTTCTGCGCTCGGCCCTTCCGGCTGGCTGGTTTATTGCTGATAAATCTGGAGCCGGTGAGCGTGGGTCTCGCGGTATCATTGCAGCACTGGGGCCAGATGGTAAGCCCTCCCGTATCGTAGTTATCTACACGACGGGGAGTCAGGCAACTATGGATGAACGAAATAGACAGATCGCTGAGATAGGTGCCTCACTGATTAAGCATTGGTAACTGTCAGACCAAGTTTACTCATATATACTTTAGATTGATTTAAAACTTCATTTTTAATTTAAAAGGATCTAGGTGAAGATCCTTTTTGATAATCTCATGACCAAAATCCCTTAACGTGAGTTTTCGTTCCACTGAGCGTCAGACCCCGTAGAAAAGATCAAAGGATCTTCTTGAGATCCTTTTTTTCTGCGCGTAATCTGCTGCTTGCAAACAAAAAAACCACCGCTACCAGCGGTGGTTTGTTTGCCGGATCAAGAGCTACCAACTCTTTTTCCGAAGGTAACTGGCTTCAGCAGAGCGCAGATACCAAATACTGTTCTTCTAGTGTAGCCGTAGTTAGGCCACCACTTCAAGAACTCTGTAGCACCGCCTACATACCTCGCTCTGCTAATCCTGTTACCAGTGGCTGCTGCCAGTGGCGATAAGTCGTGTCTTACCGGGTTGGACTCAAGACGATAGTTACCGGATAAGGCGCAGCGGTCGGGCTGAACGGGGGGTTCGTGCACACAGCCCAGCTTGGAGCGAACGACCTACACCGAACTGAGATACCTACAGCGTGAGCTATGAGAAAGCGCCACGCTTCCCGAAGGGAGAAAGGCGGACAGGTATCCGGTAAGCGGCAGGGTCGGAACAGGAGAGCGCACGAGGGAGCTTCCAGGGGGAAACGCCTGGTATCTTTATAGTCCTGTCGGGTTTCGCCACCTCTGACTTGAGCGTCGATTTTTGTGATGCTCGTCAGGGGGGCGGAGCCTATGGAAAAACGCCAGCAACGCGGCCTTTTTACGGTTCCTGGCCTTTTGCTGGCCTTTTGCTCACATGTTCTTTCCTGCGTTATCCCCTGATTCTGTGGATAACCGTATTACCGCCTTTGAGTGAGCTGATACCGCTCGCCGCAGCCGAACGACCGAGCGCAGCGAGTCAGTGAGCGAGGAAGCGGAAGAGCGCCCAATACGCAAACCGCCTCTCCCCGCGCGTTGGCCGATTCATTAATGCAGCTGGCACGACAGGTTTCCCGACTGGAAAGCGGGCAGTGAGCGCAACGCAATTAATGTGAGTTAGCTCACTCATTAGGCACCCCAGGCTTTACACTTTATGCTTCCGGCTCGTATGTTGTGTGGAATTGTGAGCGGATAACAATTTCACACAGGAAACAGCTATGACCATGATTACGCCAAGCTCTAGCTAGAGGTCGAGTCCCTCCCCAGCAGGCAGAAGTATGCAAAGCATGCATCTCAATTAGTCAGCAACCATAGTCCCGCCCCTAACTCCGCCCATCCCGCCCCTAACTCCGCCCAGTTCCGCCCATTCTCCGCCCCATGGCTGACTAATTTTTTTTATTTATGCAGAGGCCGAGGCCGCCTCGGCCTCTGAGCTATTCCAGAAGTAGTGAGGAGGCTTTTTTGGAGGCCTAGGCTTTTGCAAAAAGCTTTGCAAAGATGGATAAAGTTTTAAACAGAGAGGAATCTTTGCAGCTAATGGACCTTCTAGGTCTTGAAAGGAGTGGGAATTGGCTCCGGTGCCCGTCAGTGGGCAGAGCGCACATCGCCCACAGTCCCCGAGAAGTTGGGGGGAGGGGTCGGCAATTGAACCGGTGCCTAGAGAAGGTGGCGCGGGGTAAACTGGGAAAGTGATGTCGTGTACTGGCTCCGCCTTTTTCCCGAGGGTGGGGGAGAACCGTATATAAGTGCAGTAGTCGCCGTGAACGTTCTTTTTCGCAACGGGTTTGCCGCCAGAACACAGGTAAGTGCCGTGTGTGGTTCCCGCGGGCCTGGCCTCTTTACGGGTTATGGCCCTTGCGTGCCTTGAATTACTTCCACCTGGCTGCAGTACGTGATTCTTGATCCCGAGCTTCGGGTTGGAAGTGGGTGGGAGAGTTCGAGGCCTTGCGCTTAAGGAGCCCCTTCGCCTCGTGCTTGAGTTGAGGCCTGGCCTGGGCGCTGGGGCCGCCGCGTGCGAATCTGGTGGCACCTTCGCGCCTGTCTCGCTGCTTTCGATAAGTCTCTAGCCATTTAAAATTTTTGATGACCTGCTGCGACGCTTTTTTTCTGGCAAGATAGTCTTGTAAATGCGGGCCAAGATCTGCACACTGGTATTTCGGTTTTTGGGGCCGCGGGCGGCGACGGGGCCCGTGCGTCCCAGCGCACATGTTCGGCGAGGCGGGGCCTGCGAGCGCGGCCACCGAGAATCGGACGGGGGTAGTCTCAAGCTGGCCGGCCTGCTCTGGTGCCTGGCCTCGCGCCGCCGTGTATCGCCCCGCCCTGGGCGGCAAGGCTGGCCCGGTCGGCACCAGTTGCGTGAGCGGAAAGATGGCCGCTTCCCGGCCCTGCTGCAGGGAGCTCAAAATGGAGGACGCGGCGCTCGGGAGAGCGGGCGGGTGAGTCACCCACACAAAGGAAAAGGGCCTTTCCGTCCTCAGCCGTCGCTTCATGTGACTCCACGGAGTACCGGGCGCCGTCCAGGCACCTCGATTAGTTCTCGAGCTTTTGGAGTACGTCGTCTTTAGGTTGGGGGGAGGGGTTTTATGCGATGGAGTTTCCCCACACTGAGTGGGTGGAGACTGAAGTTAGGCCAGCTTGGCACTTGATGTAATTCTCCTTGGAATTTGCCCTTTTTGAGTTTGGATCTTGGTTCATTCTCAAGCCTCAGACAGTGGTTCAAAGTTTTTTTCTTCCATTTCAGGTGTCGTGAGGAATTCTCTAGAGATCCCTCGACCTCGAGATCCATTGTGCCCGGGCGCCACCATGGACATGCGCGTGCCCGCCCAGCTGCTGGGCCTGCTGCTGCTGTGGTTCCCCGGCTCGCGATGC 77 V7GCGTCGACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAAAGTTGAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAAGCCGCGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGCGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAATGAGCGGCCGCTCGAGGCCGGCAAGGCCGGATCCCCCGACCTCGACCTCTGGCTAATAAAGGAAATTTATTTTCATTGCAATAGTGTGTTGGAATTTTTTGTGTCTCTCACTCGGAAGGACATATGGGAGGGCAAATCATTTGGTCGAGATCCCTCGGAGATCTCTAGCTAGAGGATCGATCCCCGCCCCGGACGAACTAAACCTGACTACGACATCTCTGCCCCTTCTTCGCGGGGCAGTGCATGTAATCCCTTCAGTTGGTTGGTACAACTTGCCAACTGGGCCCTGTTCCACATGTGACACGGGGGGGGACCAAACACAAAGGGGTTCTCTGACTGTAGTTGACATCCTTATAAATGGATGTGCACATTTGCCAACACTGAGTGGCTTTCATCCTGGAGCAGACTTTGCAGTCTGTGGACTGCAACACAACATTGCCTTTATGTGTAACTCTTGGCTGAAGCTCTTACACCAATGCTGGGGGACATGTACCTCCCAGGGGCCCAGGAAGACTACGGGAGGCTACACCAACGTCAATCAGAGGGGCCTGTGTAGCTACCGATAAGCGGACCCTCAAGAGGGCATTAGCAATAGTGTTTATAAGGCCCCCTTGTTAACCCTAAACGGGTAGCATATGCTTCCCGGGTAGTAGTATATACTATCCAGACTAACCCTAATTCAATAGCATATGTTACCCAACGGGAAGCATATGCTATCGAATTAGGGTTAGTAAAAGGGTCCTAAGGAACAGCGATATCTCCCACCCCATGAGCTGTCACGGTTTTATTTACATGGGGTCAGGATTCCACGAGGGTAGTGAACCATTTTAGTCACAAGGGCAGTGGCTGAAGATCAAGGAGCGGGCAGTGAACTCTCCTGAATCTTCGCCTGCTTCTTCATTCTCCTTCGTTTAGCTAATAGAATAACTGCTGAGTTGTGAACAGTAAGGTGTATGTGAGGTGCTCGAAAACAAGGTTTCAGGTGACGCCCCCAGAATAAAATTTGGACGGGGGGTTCAGTGGTGGCATTGTGCTATGACACCAATATAACCCTCACAAACCCCTTGGGCAATAAATACTAGTGTAGGAATGAAACATTCTGAATATCTTTAACAATAGAAATCCATGGGGTGGGGACAAGCCGTAAAGACTGGATGTCCATCTCACACGAATTTATGGCTATGGGCAACACATAATCCTAGTGCAATATGATACTGGGGTTATTAAGATGTGTCCCAGGCAGGGACCAAGACAGGTGAACCATGTTGTTACACTCTATTTGTAACAAGGGGAAAGAGAGTGGACGCCGACAGCAGCGGACTCCACTGGTTGTCTCTAACACCCCCGAAAATTAAACGGGGCTCCACGCCAATGGGGCCCATAAACAAAGACAAGTGGCCACTCTTTTTTTTGAAATTGTGGAGTGGGGGCACGCGTCAGCCCCCACACGCCGCCCTGCGGTTTTGGACTGTAAAATAAGGGTGTAATAACTTGGCTGATTGTAACCCCGCTAACCACTGCGGTCAAACCACTTGCCCACAAAACCACTAATGGCACCCCGGGGAATACCTGCATAAGTAGGTGGGCGGGCCAAGATAGGGGCGCGATTGCTGCGATCTGGAGGACAAATTACACACACTTGCGCCTGAGCGCCAAGCACAGGGTTGTTGGTCCTCATATTCACGAGGTCGCTGAGAGCACGGTGGGCTAATGTTGCCATGGGTAGCATATACTACCCAAATATCTGGATAGCATATGCTATCCTAATCTATATCTGGGTAGCATAGGCTATCCTAATCTATATCTGGGTAGCATATGCTATCCTAATCTATATCTGGGTAGTATATGCTATCCTAATTTATATCTGGGTAGCATAGGCTATCCTAATCTATATCTGGGTAGCATATGCTATCCTAATCTATATCTGGGTAGTATATGCTATCCTAATCTGTATCCGGGTAGCATATGCTATCCTAATAGAGATTAGGGTAGTATATGCTATCCTAATTTATATCTGGGTAGCATATACTACCCAAATATCTGGATAGCATATGCTATCCTAATCTATATCTGGGTAGCATATGCTATCCTAATCTATATCTGGGTAGCATAGGCTATCCTAATCTATATCTGGGTAGCATATGCTATCCTAATCTATATCTGGGTAGTATATGCTATCCTAATTTATATCTGGGTAGCATAGGCTATCCTAATCTATATCTGGGTAGCATATGCTATCCTAATCTATATCTGGGTAGTATATGCTATCCTAATCTGTATCCGGGTAGCATATGCTATCCTCATGATAAGCTGTCAAACATGAGAATTTTCTTGAAGACGAAAGGGCCTCGTGATACGCCTATTTTTATAGGTTAATGTCATGATAATAATGGTTTCTTAGACGTCAGGTGGCACTTTTCGGGGAAATGTGCGCGGAACCCCTATTTGTTTATTTTTCTAAATACATTCAAATATGTATCCGCTCATGAGACAATAACCCTGATAAATGCTTCAATAATATTGAAAAAGGAAGAGTATGAGTATTCAACATTTCCGTGTCGCCCTTATTCCCTTTTTTGCGGCATTTTGCCTTCCTGTTTTTGCTCACCCAGAAACGCTGGTGAAAGTAAAAGATGCTGAAGATCAGTTGGGTGCACGAGTGGGTTACATCGAACTGGATCTCAACAGCGGTAAGATCCTTGAGAGTTTTCGCCCCGAAGAACGTTTTCCAATGATGAGCACTTTTAAAGTTCTGCTATGTGGCGCGGTATTATCCCGTGTTGACGCCGGGCAAGAGCAACTCGGTCGCCGCATACACTATTCTCAGAATGACTTGGTTGAGTACTCACCAGTCACAGAAAAGCATCTTACGGATGGCATGACAGTAAGAGAATTATGCAGTGCTGCCATAACCATGAGTGATAACACTGCGGCCAACTTACTTCTGACAACGATCGGAGGACCGAAGGAGCTAACCGCTTTTTTGCACAACATGGGGGATCATGTAACTCGCCTTGATCGTTGGGAACCGGAGCTGAATGAAGCCATACCAAACGACGAGCGTGACACCACGATGCCTGCAGCAATGGCAACAACGTTGCGCAAACTATTAACTGGCGAACTACTTACTCTAGCTTCCCGGCAACAATTAATAGACTGGATGGAGGCGGATAAAGTTGCAGGACCACTTCTGCGCTCGGCCCTTCCGGCTGGCTGGTTTATTGCTGATAAATCTGGAGCCGGTGAGCGTGGGTCTCGCGGTATCATTGCAGCACTGGGGCCAGATGGTAAGCCCTCCCGTATCGTAGTTATCTACACGACGGGGAGTCAGGCAACTATGGATGAACGAAATAGACAGATCGCTGAGATAGGTGCCTCACTGATTAAGCATTGGTAACTGTCAGACCAAGTTTACTCATATATACTTTAGATTGATTTAAAACTTCATTTTTAATTTAAAAGGATCTAGGTGAAGATCCTTTTTGATAATCTCATGACCAAAATCCCTTAACGTGAGTTTTCGTTCCACTGAGCGTCAGACCCCGTAGAAAAGATCAAAGGATCTTCTTGAGATCCTTTTTTTCTGCGCGTAATCTGCTGCTTGCAAACAAAAAAACCACCGCTACCAGCGGTGGTTTGTTTGCCGGATCAAGAGCTACCAACTCTTTTTCCGAAGGTAACTGGCTTCAGCAGAGCGCAGATACCAAATACTGTTCTTCTAGTGTAGCCGTAGTTAGGCCACCACTTCAAGAACTCTGTAGCACCGCCTACATACCTCGCTCTGCTAATCCTGTTACCAGTGGCTGCTGCCAGTGGCGATAAGTCGTGTCTTACCGGGTTGGACTCAAGACGATAGTTACCGGATAAGGCGCAGCGGTCGGGCTGAACGGGGGGTTCGTGCACACAGCCCAGCTTGGAGCGAACGACCTACACCGAACTGAGATACCTACAGCGTGAGCTATGAGAAAGCGCCACGCTTCCCGAAGGGAGAAAGGCGGACAGGTATCCGGTAAGCGGCAGGGTCGGAACAGGAGAGCGCACGAGGGAGCTTCCAGGGGGAAACGCCTGGTATCTTTATAGTCCTGTCGGGTTTCGCCACCTCTGACTTGAGCGTCGATTTTTGTGATGCTCGTCAGGGGGGCGGAGCCTATGGAAAAACGCCAGCAACGCGGCCTTTTTACGGTTCCTGGCCTTTTGCTGGCCTTTTGCTCACATGTTCTTTCCTGCGTTATCCCCTGATTCTGTGGATAACCGTATTACCGCCTTTGAGTGAGCTGATACCGCTCGCCGCAGCCGAACGACCGAGCGCAGCGAGTCAGTGAGCGAGGAAGCGGAAGAGCGCCCAATACGCAAACCGCCTCTCCCCGCGCGTTGGCCGATTCATTAATGCAGCTGGCACGACAGGTTTCCCGACTGGAAAGCGGGCAGTGAGCGCAACGCAATTAATGTGAGTTAGCTCACTCATTAGGCACCCCAGGCTTTACACTTTATGCTTCCGGCTCGTATGTTGTGTGGAATTGTGAGCGGATAACAATTTCACACAGGAAACAGCTATGACCATGATTACGCCAAGCTCTAGCTAGAGGTCGAGTCCCTCCCCAGCAGGCAGAAGTATGCAAAGCATGCATCTCAATTAGTCAGCAACCATAGTCCCGCCCCTAACTCCGCCCATCCCGCCCCTAACTCCGCCCAGTTCCGCCCATTCTCCGCCCCATGGCTGACTAATTTTTTTTATTTATGCAGAGGCCGAGGCCGCCTCGGCCTCTGAGCTATTCCAGAAGTAGTGAGGAGGCTTTTTTGGAGGCCTAGGCTTTTGCAAAAAGCTTTGCAAAGATGGATAAAGTTTTAAACAGAGAGGAATCTTTGCAGCTAATGGACCTTCTAGGTCTTGAAAGGAGTGGGAATTGGCTCCGGTGCCCGTCAGTGGGCAGAGCGCACATCGCCCACAGTCCCCGAGAAGTTGGGGGGAGGGGTCGGCAATTGAACCGGTGCCTAGAGAAGGTGGCGCGGGGTAAACTGGGAAAGTGATGTCGTGTACTGGCTCCGCCTTTTTCCCGAGGGTGGGGGAGAACCGTATATAAGTGCAGTAGTCGCCGTGAACGTTCTTTTTCGCAACGGGTTTGCCGCCAGAACACAGGTAAGTGCCGTGTGTGGTTCCCGCGGGCCTGGCCTCTTTACGGGTTATGGCCCTTGCGTGCCTTGAATTACTTCCACCTGGCTGCAGTACGTGATTCTTGATCCCGAGCTTCGGGTTGGAAGTGGGTGGGAGAGTTCGAGGCCTTGCGCTTAAGGAGCCCCTTCGCCTCGTGCTTGAGTTGAGGCCTGGCCTGGGCGCTGGGGCCGCCGCGTGCGAATCTGGTGGCACCTTCGCGCCTGTCTCGCTGCTTTCGATAAGTCTCTAGCCATTTAAAATTTTTGATGACCTGCTGCGACGCTTTTTTTCTGGCAAGATAGTCTTGTAAATGCGGGCCAAGATCTGCACACTGGTATTTCGGTTTTTGGGGCCGCGGGCGGCGACGGGGCCCGTGCGTCCCAGCGCACATGTTCGGCGAGGCGGGGCCTGCGAGCGCGGCCACCGAGAATCGGACGGGGGTAGTCTCAAGCTGGCCGGCCTGCTCTGGTGCCTGGCCTCGCGCCGCCGTGTATCGCCCCGCCCTGGGCGGCAAGGCTGGCCCGGTCGGCACCAGTTGCGTGAGCGGAAAGATGGCCGCTTCCCGGCCCTGCTGCAGGGAGCTCAAAATGGAGGACGCGGCGCTCGGGAGAGCGGGCGGGTGAGTCACCCACACAAAGGAAAAGGGCCTTTCCGTCCTCAGCCGTCGCTTCATGTGACTCCACGGAGTACCGGGCGCCGTCCAGGCACCTCGATTAGTTCTCGAGCTTTTGGAGTACGTCGTCTTTAGGTTGGGGGGAGGGGTTTTATGCGATGGAGTTTCCCCACACTGAGTGGGTGGAGACTGAAGTTAGGCCAGCTTGGCACTTGATGTAATTCTCCTTGGAATTTGCCCTTTTTGAGTTTGGATCTTGGTTCATTCTCAAGCCTCAGACAGTGGTTCAAAGTTTTTTTCTTCCATTTCAGGTGTCGTGAGGAATTCTCTAGAGATCCCTCGACCTCGAGATCCATTGTGCCCGGGCGCCACCATGGAGTTTGGGCTGAGCTGGCTTTTTCTTGTCGCGATTTTAAAAGGTGTCCAGTGC

The present invention incorporates by reference in their entiretytechniques well known in the field of molecular biology and drugdelivery. These techniques include, but are not limited to, techniquesdescribed in the following publications:

-   Ausubel et al. (eds.), Current Protocols in Molecular Biology, John    Wiley &Sons, NY (1993);-   Ausubel, F. M. et al. eds., Short Protocols In Molecular Biology    (4th Ed. 1999) John Wiley & Sons, NY. (ISBN 0-471-32938-X).-   Controlled Drug Bioavailability, Drug Product Design and    Performance, Smolen and Ball (eds.), Wiley, New York (1984);-   Giege, R. and Ducruix, A. Barrett, Crystallization of Nucleic Acids    and Proteins, a Practical Approach, 2nd ea., pp. 20 1-16, Oxford    University Press, New York, N.Y., (1999);-   Goodson, in Medical Applications of Controlled Release, vol. 2, pp.    115-138 (1984);-   Hammerling, et al., in: Monoclonal Antibodies and T-Cell Hybridomas    563-681 (Elsevier, N.Y., 1981;-   Harlow et al., Antibodies: A Laboratory Manual, (Cold Spring Harbor    Laboratory Press, 2nd ed. 1988);-   Kabat et al., Sequences of Proteins of Immunological Interest    (National Institutes of Health, Bethesda, Md. (1987) and (1991);-   Kabat, E. A., et al. (1991) Sequences of Proteins of Immunological    Interest, Fifth Edition, U.S. Department of Health and Human    Services, NIH Publication No. 91-3242;-   Kontermann and Dubel eds., Antibody Engineering (2001)    Springer-Verlag. New York. 790 pp. (ISBN 3-540-41354-5).-   Kriegler, Gene Transfer and Expression, A Laboratory Manual,    Stockton Press, NY (1990);-   Lu and Weiner eds., Cloning and Expression Vectors for Gene Function    Analysis (2001) BioTechniques Press. Westborough, Mass. 298 pp.    (ISBN 1-881299-21-X).-   Medical Applications of Controlled Release, Langer and Wise (eds.),    CRC Pres., Boca Raton, Fla. (1974);-   Old, R. W. & S. B. Primrose, Principles of Gene Manipulation: An    Introduction To Genetic Engineering (3d Ed. 1985) Blackwell    Scientific Publications, Boston. Studies in Microbiology; V.2:409    pp. (ISBN 0-632-01318-4).-   Sambrook, J. et al. eds., Molecular Cloning: A Laboratory Manual (2d    Ed. 1989) Cold Spring Harbor Laboratory Press, NY. Vols. 1-3. (ISBN    0-87969-309-6).-   Sustained and Controlled Release Drug Delivery Systems, J. R.    Robinson, ed., Marcel Dekker, Inc., New York, 1978-   Winnacker, E. L. From Genes To Clones: Introduction To Gene    Technology (1987) VCH Publishers, NY (translated by Horst    Ibelgaufts). 634 pp. (ISBN 0-89573-614-4).

INCORPORATION BY REFERENCE

The contents of all cited references (including literature references,patents, patent applications, and websites) that may be cited throughoutthis application are hereby expressly incorporated by reference in theirentirety for any purpose, as are the references cited therein. Thepractice of the present invention will employ, unless otherwiseindicated, conventional techniques of immunology, molecular biology andcell biology, which are well known in the art.

EQUIVALENTS

The invention may be embodied in other specific forms without departingfrom the spirit or essential characteristics thereof. The foregoingembodiments are therefore to be considered in all respects illustrativerather than limiting of the invention described herein. Scope of theinvention is thus indicated by the appended claims rather than by theforegoing description, and all changes that come within the meaning andrange of equivalency of the claims are therefore intended to be embracedherein.

We claim:
 1. A binding protein comprising first and second polypeptidechains, wherein the first polypeptide chain comprises a firstVD1-(X1)n-VD2-C-(X2)n, wherein VD1 is a first heavy chain variabledomain; VD2 is a second heavy chain variable domain; C is a heavy chainconstant domain; X1 is a linker; X2 is an Fc region; and wherein thesecond polypeptide chain comprises a second VD1-(X1)n-VD2-C-(X2)n,wherein VD1 is a first light chain variable domain; VD2 is a secondlight chain variable domain; C is a light chain constant domain; X1 is alinker; X2 does not comprise an Fc region; n is 0 or 1, and wherein thebinding protein is capable of binding a pair of antigens selected fromthe group consisting of IL-1alpha and IL-1beta, wherein either the VD1heavy and light chain variable domain or the VD2 heavy and light chainvariable domain form an antigen binding domain that binds the IL-1betaantigen and comprises the amino acid sequences of SEQ ID NOs: 30 and 31or SEQ ID NOs: 32 and 33, and wherein the other of the VD1 or VD2 heavyand light chain variable domains forms an antigen binding domain thatbinds the IL-1alpha antigen.
 2. The binding protein of claim 1, whereinthe heavy chain variable domain of the antigen binding domain that bindsthe IL-1alpha antigen comprises an amino acid sequence selected from thegroup consisting of SEQ ID NOs: 40, 42, 44, and 46, and wherein thelight chain variable domain of the antigen binding domain that binds theIL-1alpha antigen comprises an amino acid sequence selected from thegroup consisting of SEQ ID NOs: 41, 43, 45, and
 47. 3. The bindingprotein of claim 1, wherein X1 or X2 is an amino acid sequence selectedfrom the group consisting of SEQ ID NOs: 1-29.
 4. The binding protein ofclaim 1, wherein the binding protein comprises two first polypeptidechains and two second polypeptide chains.
 5. The binding protein ofclaim 1, wherein the Fc region is a variant sequence Fc region.
 6. Abinding protein capable of binding two antigens comprising fourpolypeptide chains, wherein two polypeptide chains compriseVD1-(X1)n-VD2-C-(X2)n, wherein VD1 is a first heavy chain variabledomain; VD2 is a second heavy chain variable domain; C is a heavy chainconstant domain; X1 is a linker; X2 is an Fc region; and wherein twopolypeptide chains comprise VD1-(X1)n-VD2-C-(X2)n, wherein VD1 is afirst light chain variable domain; VD2 is a second light chain variabledomain; C is a light chain constant domain; X1 is a linker; X2 does notcomprise an Fc region; n is 0 or 1; and wherein the VD1 heavy and lightchain variable domains form an antigen binding domain that binds theIL-1beta antigen and comprise the amino acid sequences of SEQ ID NOs 30and 31 or SEQ ID NOs 32 and 33; and wherein the VD2 heavy and lightchain variable domains forms an antigen binding domain that binds theIL-1 alpha antigen.
 7. A binding protein capable of binding two antigenscomprising four polypeptide chains, wherein two polypeptide chainscomprise VD1-(X1)n-VD2-C-(X2)n, wherein VD1 is a first heavy chainvariable domain; VD2 is a second heavy chain variable domain; C is aheavy chain constant domain; X1 is a linker; X2 is an Fc region; n is 0or 1; and wherein two polypeptide chains comprise VD1-(X1)n-VD2-C-(X2)n,wherein VD1 is a first light chain variable domain; VD2 is a secondlight chain variable domain; C is a light chain constant domain; X1 is alinker; X2 does not comprise an Fc region; n is 0 or 1; and wherein theVD2 heavy and light chain variable domains form an antigen bindingdomain that binds the IL-1beta antigen and comprise the amino acidsequences of SEQ ID NOs 30 and 31 or SEQ ID NOs 32 and 33; and whereinthe VD1 heavy and light chain variable domains forms an antigen bindingdomain that binds the IL-1 alpha antigen.
 8. The binding protein of anyone of claims 1, 6, and 7, wherein the binding protein has an on rateconstant (Kon) to IL-1beta antigen selected from the group consistingof: at least about 10²M⁻¹s⁻¹; at least about 10³M⁻¹s⁻¹; at least about10⁴M⁻¹s⁻¹; at least about 10⁵M⁻¹s⁻¹; and at least about 10⁶M⁻¹s⁻¹, asmeasured by surface plasmon resonance.
 9. The binding protein of any oneof claims 1, 6, and 7, wherein the binding protein has an off rateconstant (Koff) to IL-1beta antigen selected from the group consistingof: at most about 10⁻³s⁻¹; at most about 10⁻⁴s⁻¹; at most about 10⁻⁵s⁻¹;and at most about 10⁻⁶s⁻¹, as measured by surface plasmon resonance. 10.The binding protein of any one of claims 1, 6, and 7, wherein thebinding protein has a dissociation constant (K_(D)) to IL-1beta antigenselected from the group consisting of: at most about 10⁻⁷ M; at mostabout 10⁻⁸ M; at most about 10⁻⁹ M; at most about 10⁻¹⁰ M; at most about10⁻¹¹ M; at most about 10⁻¹² M; and at most 10⁻¹³M.
 11. A bindingprotein conjugate comprising a binding protein of any one of claims 1,6, and 7, the binding protein conjugate further comprising an agentselected from the group consisting of an immunoadhesion molecule, animaging agent, a therapeutic agent, and a cytotoxic agent.
 12. Thebinding protein conjugate of claim 11, wherein the imaging agent isselected from the group consisting of a radiolabel, an enzyme, afluorescent label, a luminescent label, a bioluminescent label, amagnetic label, and biotin.
 13. The binding protein conjugate of claim12, wherein the imaging agent is a radiolabel selected from the groupconsisting of: ³H, ¹⁴C, ³⁵S, ⁹⁰Y, ⁹⁹Tc, ¹¹¹In, ¹²⁵I, ¹³¹I, ¹⁷⁷Lu, ¹⁶⁶Ho,and ¹⁵³Sm.
 14. The binding protein conjugate of claim 11, wherein theagent is a therapeutic or cytotoxic agent selected from the groupconsisting of an anti-metabolite, an alkylating agent, an antibiotic, agrowth factor, a cytokine, an anti-angiogenic agent, an anti-mitoticagent, an anthracycline, toxin, and an apoptotic agent.
 15. Apharmaceutical composition comprising the binding protein of any one ofclaims 1, 6, and 7, and a pharmaceutically acceptable carrier.
 16. Thepharmaceutical composition of claim 15, wherein the composition furthercomprises at least one additional therapeutic agent.
 17. Thepharmaceutical composition of claim 16, wherein the additionaltherapeutic agent is selected from the group consisting of: a cytotoxicagent, an angiogenesis inhibitor, a kinase inhibitor, a co-stimulationmolecule blocker; an adhesion molecule blocker, an anti-cytokineantibody or functional fragment thereof, methotrexate, cyclosporin,rapamycin, FK506, a detectable label or reporter, a TNF antagonist, anantirheumatic, a muscle relaxant, a narcotic, a non-steroidanti-inflammatory drug (NSAID), an analgesic, an anesthetic, a sedative,a local anesthetic, a neuromuscular blocker, an antimicrobial, anantipsoriatic, a corticosteriod, an anabolic steroid, an erythropoietin,an immunization, an immunoglobulin, an immunosuppressive, a growthhormone, a hormone replacement drug, a radiopharmaceutical, anantidepressant, an antipsychotic, a stimulant, an asthma medication, abeta agonist, an inhaled steroid, an epinephrine or analog, a cytokine,and a cytokine antagonist.
 18. The binding protein of claim 1, whereinthe VD1 or VD2 heavy and light chain variable domains comprise the aminoacid sequences of SEQ ID NOs 30 and
 31. 19. The binding protein of claim1, wherein the VD1 or VD2 heavy and light chain variable domainscomprise the amino acid sequences of SEQ ID NOs 32 and
 33. 20. Thebinding protein of claim 6, wherein the VD1 heavy and light chainvariable domains comprise the amino acid sequences of SEQ ID NOs 30 and31.
 21. The binding protein of claim 6, wherein the VD1 heavy and lightchain variable domains comprise the amino acid sequences of SEQ ID NOs32 and
 33. 22. The binding protein of claim 6, wherein the VD2 heavychain variable domain comprises an amino acid sequence selected from thegroup consisting of SEQ ID NOs: 40, 42, 44, and 46, and wherein the VD2light chain variable domain comprises an amino acid sequence selectedfrom the group consisting of SEQ ID NOs: 41, 43, 45, and
 47. 23. Thebinding protein of claim 7, wherein the VD2 heavy and light chainvariable domains comprises the amino acid sequences of SEQ ID NOs 30 and31.
 24. The binding protein of claim 7, wherein the VD2 heavy and lightchain variable domains comprises the amino acid sequences of SEQ ID NOs32 and
 33. 25. The binding protein of claim 1, wherein the VD1 heavy andlight chain variable domains comprise the amino acid sequences of SEQ IDNOs 30 and
 31. 26. The binding protein of claim 1, wherein the VD1 heavyand light chain variable domains comprise the amino acid sequences ofSEQ ID NOs 32 and
 33. 27. The binding protein of claim 1, wherein theVD2 heavy and light chain variable domains comprise the amino acidsequences of SEQ ID NOs 30 and
 31. 28. The binding protein of claim 1,wherein the VD2 heavy and light chain variable domains comprise theamino acid sequences of SEQ ID NOs 32 and 33.